Recombinant proteins that simultaneously bind HGF, VEGF-A and serum albumin, comprising ankyrin repeat domains

ABSTRACT

New designed ankyrin repeat domains with binding specificity for serum albumin, recombinant binding proteins comprising at least two designed ankyrin repeat domains with binding specificity for serum albumin, as well as recombinant binding proteins comprising at least one designed ankyrin repeat domain with binding specificity for hepatocyte growth factor (HGF), at least one designed ankyrin repeat domain with binding specificity for vascular endothelial growth factor (VEGF-A), and at least two designed ankyrin repeat domain with binding specificity for serum albumin are described, as well as nucleic acids encoding such designed ankyrin repeat domains and recombinant binding proteins, pharmaceutical compositions comprising such designed ankyrin repeat domains, recombinant binding proteins or nucleic acids and the use of such designed ankyrin repeat domains, recombinant binding proteins, nucleic acids or pharmaceutical compositions in the treatment of diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of and the priority toEuropean patent application EP 15162502, filed Apr. 2, 2015, with theEuropean Patent Office. The present application also claims the benefitof and the priority to European patent application EP 15162511, filedApr. 2, 2015, with the European Patent Office. The contents of Europeanpatent applications EP 15162502 and EP 15162511 are incorporated hereinby reference for all purposes in their entireties.

FIELD OF THE DISCLOSURE

Provided is a new designed ankyrin repeat domain with bindingspecificity for serum albumin exhibiting improved storage stabilityproperties. Provided are also recombinant binding proteins comprising atleast two designed ankyrin repeat domains with binding specificity forserum albumin, which exhibit improved pharmacokinetic propertiescompared to the recombinant binding proteins comprising only onedesigned ankyrin repeat domain with binding specificity for serumalbumin. Particularly provided are recombinant binding proteinscomprising at least one designed ankyrin repeat domain with bindingspecificity for hepatocyte growth factor (HGF), comprising at least onedesigned ankyrin repeat domain with binding specificity for vascularendothelial growth factor A (VEGF-A), and comprising at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin. Furthermore provided are nucleic acids encoding such designedankyrin repeat domains and/or recombinant binding proteins,pharmaceutical compositions comprising such designed ankyrin repeatdomains, recombinant binding proteins or nucleic acids, and the use ofsuch designed ankyrin repeat domains, recombinant binding proteins,nucleic acids, or pharmaceutical compositions in the treatment of adisease.

BACKGROUND

The following discussion of the background is merely provided to aid thereader in understanding the invention and is not admitted to describe orconstitute prior art to the present invention.

There are, beside antibodies, novel binding proteins or binding domainsthat can be used to specifically bind a target molecule (e.g. Binz, H.K., Amstutz, P., Plückthun, A., Nat. Biotechnol. 23, 1257-1268, 2005).One such novel class of binding proteins or binding domains notpossessing an Fc are based on designed repeat proteins or designedrepeat domains, such as designed ankyrin repeat proteins or designedankyrin repeat domains (WO 2002/020565; Binz, H. K., Amstutz, P., Kohl,A., Stumpp, M. T., Briand, C., Forrer, P., Gruner, M. G., Pluckthun, A.,Nat. Biotechnol. 22, 575-582, 2004). WO 2002/020565 describes how largelibraries of repeat proteins, such as ankyrin repeat proteins, can beconstructed, and their general application. WO 2012/069654 describesrecombinant binding proteins comprising a designed ankyrin repeat domainwith binding specificity for serum albumin. WO 2010/060748 describesrecombinant binding proteins comprising designed ankyrin repeat domainswith binding specificity for VEGF-A, and WO 2011/135067 describesmodified versions of such recombinant binding proteins specific forbinding to VEGF-A. WO 2014/191574 describes recombinant binding proteinscomprising designed ankyrin repeat domains with binding specificity forHGF. None of these patent applications discloses a recombinant bindingprotein comprising a designed ankyrin repeat domain with bindingspecificity for VEGF-A and a designed ankyrin repeat domain with bindingspecificity for HGF.

Unlike e.g. IgG antibodies, which exhibit long systemic half-livesmediated by FcRn recycling, proteins comprising designed ankyrin repeatdomains typically exhibit a fast pharmacokinetic clearance and shortterminal half-lives, unless the protein comprises elements that improvethe pharmacokinetic properties, such as e.g. a designed ankyrin repeatdomain with binding specificity to serum albumin described in WO2012/069654. Using serum albumin binding for improving pharmacokineticproperties of proteins is a process well-known in the art (see e.g. WO9101743; Frejd F. Y., 2012 (in Kontermann, R (Ed.) “Therapeuticproteins: strategies to modulate their plasma half-lives”, Wiley-VCHVerlag GmbH, 2012, ISBN 978-3-527-32849-9); Nguyen, A., Reyes, A. E.II., Zhang, M., McDonald, P., Wong, W. L., Damico, L. A., Dennis, M. S.Protein Eng. Des. Sel. 19, 291-297, 2006; WO 2008/096158; WO2006/122787; WO 2011/095545; and WO 2012/069654). In order to be able touse designed ankyrin repeat domains with binding specificity for serumalbumin in clinical drug candidates, the storage stability of knowndesigned ankyrin repeat domains with binding specificity for serumalbumin has to be improved. Disclosed herein are designed ankyrin repeatdomains with binding specificity for serum albumin with improvedproperties.

The effect of valency of designed ankyrin repeat domains with bindingspecificity for serum albumin on the pharmacokinetic properties ofrecombinant binding proteins has not been investigated. Based onfindings of the albumin binding domain (Hopp, J., Horning, N., Zettlitz,K. A., Schwarz, A., Fuss, N., Müller, D., Kontermann, R. E. Protein Eng.Des. Sel. 23, 827-834, 2010), one skilled in the art would expect that arecombinant binding protein comprising two albumin binding proteindomains such as designed ankyrin repeat domains with binding specificityfor serum albumin would not have improved pharmacokinetic propertiescompared to a recombinant binding protein comprising only one designedankyrin repeat domain with binding specificity for serum albumin.Surprisingly, we found that this is not the case. Disclosed are thusrecombinant binding proteins comprising at least two designed ankyrinrepeat domains with binding specificity for serum albumin that exhibitimproved pharmacokinetic properties (i.e. prolonged terminal half-lives,increased exposures, reduced clearance, and/or increased percentages ofinjected dose) compared to recombinant binding proteins comprising onlyone designed ankyrin repeat domain with binding specificity for serumalbumin.

Neovascularisation (new blood vessel formation) is widely known to playan important role in the development and maintenance of tumors (Ferrera,N., and Kerbel, R. S., Nature 438, 967-974, 2005). Accordingly, theinhibition of angiogenesis has become a main cornerstone in modernclinical oncology; especially the targeting of vascular endothelialgrowth factor (VEGF) and its receptors (Hurwitz, H., Clin. ColorectalCancer, Suppl. 2, 62-68, 2004; Escudier, B., Clin. Adv. Hematol. Oncol.5, 530-531, 2007). The mammalian VEGF family consists of fiveglycoproteins referred to as VEGF-A, VEGF-B, VEGF-C, VEGF-D (also knownas FIGF) and placenta growth factor (PIGF, also known as PGF). VEGF-Ahas been shown to be an effective target for anti-angiogenic therapy(Weis, S. M., and Cheresh, D. A., Nat. Med. 17, 1359-1370, 2011). TheVEGF-A ligands bind to and activate three structurally similar type IIIreceptor tyrosine kinases, designated VEGFR-1 (also known as FLT1),VEGFR-2 (also known as KDR) and VEGFR-3 (also known as FLT4). Severalangiogenesis inhibitors have received regulatory approval to dateshowing a prolonged progression-free survival (PFS) and/or overallsurvival in various cancer types in combination with chemotherapy.Unfortunately, resistance inevitably occurs during the course oftreatment with VEGF/VEGFR inhibitors, such as the VEGF-A inhibitorbevacizumab (Avastin®), suggesting that concomitant inhibition ofadditional targets and resistance pathways may be necessary to achievesuperior clinical results (Kerbel, R. S., N. Engl. J. Med. 358,2039-2049, 2008; Hurwitz, 2004, loc. cit.; Escudier, 2007, loc. cit.).cMet tyrosine kinase is a cell surface receptor for hepatocyte growthfactor (HGF, also known as scatter factor, SF) primarily expressed onepithelial cells (Comoglio, P. M., Giordano, S., and Trusolino, L., Nat.Rev. Drug Discov. 7, 504-516, 2008). While cMet and HGF are expressed atlow levels in normal adult tissues, their expression is frequently upregulated in a broad range of human tumors, which has been correlated inpreclinical models with tumor cell survival, growth, angiogenesis,invasion and metastasis (Rong, S., Segal, S., Anver, M., Resau, J. H.,Vande Woude, G. F., Proc. Natl. Acad. Sci. USA 91, 4731-4735, 1994;Michieli, P., Mazzone, M., Basilico, C., Cavassa, S., Sottile, A.,Naldini, L., Comoglio, P. M., Cancer Cell 6, 61-73, 2004). Up-regulationof HGF and/or cMet expression and signaling has been found to beassociated with poor prognosis and drug resistance in many tumor typesin the clinic (Fasolo, A., Sessa, C., Gianni, L., Broggini, M., Ann.Oncol. 24, 14-20, 2013). Altogether this indicates that the HGF-cMetaxis is an important target for therapeutic intervention (Comoglio, 2008loc. cit.; Fasolo et al., 2013, loc. cit.). Through binding to itsreceptor, HGF mediates a number of cellular responses, includingscattering of various cell types, the formation of tubules and lumens,epithelial-mesenchymal transition, angiogenesis, liver regeneration,wound healing and embryological development. The HGF/c-Met signalingpathway has also been shown to play a role in various diseases,including many human solid tumors, in which it participates in tumordevelopment, invasion and metastasis. Current HGF/cMet pathwayinhibitors in phase II or III clinical development comprise monoclonalantibodies (mAbs) targeting the extracellular domain of cMet (i.e.MetMab from Genentech-Roche) or small molecule inhibitors of itsintracellular kinase domain. Small molecule inhibitors such astivantinib (ArQule®) and cabozantinib (Cometriq®) are very potent butless specific than mAbs and bear the potential for higher toxicity.Biological agents against HGF/SF include rilotumumab (AMG102), ahumanised mAb against HGF, and ficlatuzumab (AV-299), a humanisedanti-HGF IgG1. The use of HGF/cMet inhibitors in combination with othertargeted agents is an active field of investigation which aims tosimultaneously inhibit various signaling pathways that have redundant orsynergistic tumor functions. HGF/cMet triggers potent angiogenic signalsthat act synergistically with VEGF in inducing new tumor blood vesselsand can induce resistance to anti-angiogenic therapy such as Avastin®and Sutent® (sunitinib) in glioblastoma (Jahangiri, A., De Lay, M.,Miller, L. M., Carbonell, W. S., Hu, Y. L., Lu, K., Tom, M. W.,Paquette, J., Tokuyasu, T. A., Tsao, S., Marshall, R., Perry, A.,Bjorgan, K. M., Chaumeil, M. M., Ronen, S. M., Bergers, G., Aghi, M. K.,Clin. Cancer Res. 19, 1773-1783, 2013) and renal cell cancer,respectively.

There are currently a number of anti-HGF/cMet compounds underinvestigation in combination with other targeted agents such asanti-VEGF receptor inhibitors, which have demonstrated a favorablesafety profile in a variety of tumor types (Sharma, P. S., Sharma, R.,Tyagi, T. Curr. Cancer Drug Targets. 11, 624-653, 2011). However, suchcombination therapy approaches imply that the patient must receive twoseparate treatments, each with a different safety profile, which maylead to increased undesirable toxicities, which in turn may limit themedical treatment options. Furthermore, different treatments may besubjected to different administration schemes, which could make thedosing more burdensome for the patient. Last but not least, the dosingof various agents simultaneously may significantly increase the costsassociated to treatment and patient care.

One commercially available drug with dual cMet and VEGF inhibitoryactivity is cabozantinib (Cometriq®; a small molecule drug), an oral,multi-specific tyrosine kinase inhibitor targeting cMet and VEGFR 1-3(in addition to RET, KIT, AXL and FLT3). Cabozantinib has validated theclinical approach of simultaneously inhibiting HGF and VEGF in tumorswith a single agent (Yakes, F. M., Chen, J., Tan, J., Yamaguchi, K.,Shi, Y., Yu, P., Qian, F., Chu, F., Bentzien, F., Cancilla, B., Orf, J.,You, A., Laird, A. D., Engst, S., Lee, L., Lesch, J., Chou, Y. C., Joly,A. H., Mol. Cancer Ther. 10, 2298-2308, 2011; Castellone, M. D.,Carlomagno, F., Salvatore, G., Santoro, M., Best Pract. Res. Clin.Endocrinol. Metab. 22, 1023-1038, 2008). For instance, in castrationresistant prostate cancer, an indication where the anti-HGF mAbrilotumumab failed to demonstrate efficacy as single agent in phase IIstudies, cabozantinib showed anti-tumor activity in a high percentage ofpatients in phase II (Smith, D. C., Smith, M. R., Sweeney, C., Elfiky,A. A., Logothetis, C., Corn, P. G., Vogelzang, N. J., Small, E. J.,Harzstark, A. L., Gordon, M. S., Vaishampayan, U. N., Haas, N. B.,Spira, A. I., Lara, P. N. Jr., Lin, C. C., Srinivas, S., Sella, A.,Schöffski, P., Scheffold, C., Weitzman, A. L., Hussain, M., J. Clin.Oncol. 31, 412-419, 2013). However, activity was paralleled with a highincidence of adverse events that led to dose reductions in 62% ofpatients, raising doubts on the safety and tolerability of suchpleiotropic modes of action.

Simultaneous targeting of VEGF-A and HGF/cMet may beneficially disruptangiogenesis and tumor progression. As described hereinbefore, currenttherapies acting simultaneously on the VEGF-A/VEGFR-2 and theHGF/cMet-pathways either are based on single therapeutics that areunspecific and lead to safety findings, or involve several specifictherapeutics that have to be combined, resulting in a need ofco-administration or multiple administrations. Furthermore, some of thecurrent drugs exhibit short systemic half-lives. Thus, there is a needto provide improved drugs blocking the VEGF-A/VEGFR-2 and the HGF/cMetpathways. This is technically difficult to achieve with antibody drugs,which further suffer from the need of laborious production in mammaliancells. Provided herein are recombinant binding proteins that addressthese issues. In some embodiments a recombinant binding protein providedherein comprises at least one designed ankyrin repeat domain withbinding specificity for VEGF-A, at least one designed ankyrin repeatdomain with binding specificity for HGF, and, for pharmacokineticproperty improvement, at least two designed ankyrin repeat domains withbinding specificity for serum albumin.

SUMMARY

The present invention relates to a new designed ankyrin repeat domainwith binding specificity for serum albumin comprising the amino acidsequence of SEQ ID NO: 50, which exhibits improved storage stabilityover known designed ankyrin repeat domains with binding specificity forserum albumin. In one embodiment, the invention relates to a recombinantbinding protein comprising at least two designed ankyrin repeat domainswith binding specificity for serum albumin, wherein said designedankyrin repeat domains with binding specificity for serum albumin eachcomprise SEQ ID NO: 50. In one embodiment, the invention relates to arecombinant binding protein comprising a first, a second, a third, and afourth designed ankyrin repeat domain, wherein said first designedankyrin repeat domain has binding specificity for VEGF-A, and whereinsaid second designed ankyrin repeat domain has binding specificity forHGF, and wherein said third and fourth designed ankyrin repeat domainseach have binding specificity for serum albumin and comprise the aminoacid sequence of SEQ ID NO: 50. In one embodiment, said first, second,third and fourth designed ankyrin repeat domains of said recombinantbinding protein are in the order third-second-first-fourth from Nterminus to C terminus. In one embodiment, said first designed ankyrinrepeat domain of said recombinant binding protein comprises an aminoacid sequence selected from the group consisting of amino acid sequencesSEQ ID NOs: 12 to 21 and amino acid sequences in which up to 10 aminoacids of SEQ ID NOs: 12 to 21 are exchanged by any amino acid, and saidsecond designed ankyrin repeat domain of said recombinant bindingprotein comprises an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 23 to 37 and amino acidsequences in which up to 10 amino acids of SEQ ID NOs: 23 to 37 areexchanged by any amino acid, and said third and fourth designed ankyrinrepeat domains of said recombinant binding protein each comprise aminoacid sequence SEQ ID NO: 50, and said designed ankyrin repeat domainsare linked by polypeptide linkers comprising amino acid sequencesselected from the group consisting of amino acid sequences SEQ ID NOs: 2to 9 and amino acid sequences in which up to 4 amino acids of SEQ IDNOs: 2 to 9 are exchanged by any amino acid. In one embodiment, saidfirst designed ankyrin repeat domain of said recombinant binding proteincomprises amino acid sequences SEQ ID NO: 18, and said second designedankyrin repeat domain of said recombinant binding protein comprisesamino acid sequence SEQ ID NO: 26, and said third and fourth designedankyrin repeat domains of said recombinant binding protein each compriseamino acid sequence SEQ ID NO: 50, and said designed ankyrin repeatdomains are linked by polypeptide linkers consisting of amino acidsequence SEQ ID NO: 9. In one embodiment, the invention relates to arecombinant binding protein comprising an amino acid sequence that hasat least 90% amino acid sequence identity with the amino acid sequenceof SEQ ID NO: 134. In one embodiment, the invention relates to arecombinant binding protein comprising the amino acid sequenceconsisting of the amino acid sequence of SEQ ID NO: 134. In a preferredembodiment, the invention relates to a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134.

The invention further relates to a nucleic acid encoding the amino acidsequence of a designed ankyrin repeat domain or a recombinant bindingprotein of the invention.

The invention also relates to a pharmaceutical composition comprising arecombinant binding protein and/or a designed ankyrin repeat domain or anucleic acid of the present invention, and optionally a pharmaceuticalacceptable carrier and/or diluent.

The invention also relates to the use of the pharmaceutical compositionof the invention for the treatment of a disease. In one embodiment, itrelates to the use of the pharmaceutical composition of the inventionfor the treatment of cancer, gastric cancer, or renal cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1A shows an illustration of a designed ankyrin repeatdomain with binding specificity for serum albumin. Examples of suchankyrin repeat domains are designed ankyrin repeat domains with an aminoacid sequence selected from the group consisting of SEQ ID NOs: 40 to56, in particular the designed ankyrin repeat domain with amino acidsequence of SEQ ID NO: 50.

FIG. 1B. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1B shows an illustration of a designed ankyrin repeatdomain with binding specificity for another target than serum albumin.Examples of such ankyrin repeat domains are designed ankyrin repeatdomains with an amino acid sequence selected from the group consistingof SEQ ID NOs: 12 to 39.

FIG. 1C. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1C shows an illustration of a polypeptide linker (forexample a polypeptide with an amino acid sequence corresponding to anyof SEQ ID NOs: 2 to 9).

FIG. 1D. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1D shows an illustration of an N-terminal amino acidsequence. Examples for such N-terminal amino acid sequences are forexample the sequences MGS or GS, or polypeptide tags, as exemplified bythe amino acid sequence corresponding to SEQ ID NO: 1.

FIG. 1E. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1E shows an illustration of a bioactive compound. Such amoiety can for example be a protein or protein domain with e.g.agonistic (e.g. hormone, or enzyme), antagonistic (e.g. receptor domainor antibody fragment), or toxic (e.g. toxin) activity. Such a moiety canfor example also be a small molecule compound exhibiting e.g. agonistic,antagonistic, or toxic activity.

FIG. 1F. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1F. shows an illustration of a recombinant binding proteinprovided herein comprising two designed ankyrin repeat domains withbinding specificity for serum albumin, and one designed ankyrin repeatdomain with binding specificity for another target than serum albumin,linked by polypeptide linkers and having an N-terminal amino acidsequence. For example a recombinant binding protein with an amino acidsequence corresponding to any of SEQ ID NOs: 73 to 81 consist of suchthree designed ankyrin repeat domains, wherein SEQ ID NOs: 73, 75, 78,and 80 have the two designed ankyrin repeat domains with bindingspecificity for serum albumin flanking the respective third designedankyrin repeat domain as shown in the illustration.

FIG. 1G. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1G. shows an illustration of a recombinant binding proteinprovided herein comprising two designed ankyrin repeat domains withbinding specificity for serum albumin, and two designed ankyrin repeatdomains with binding specificities for other targets than serum albumin,linked by polypeptide linkers and having an N-terminal amino acidsequence. The two designed ankyrin repeat domains with bindingspecificity for serum albumin are flanking the two other designedankyrin repeat domains. For example a recombinant binding protein withan amino acid sequence corresponding to any of SEQ ID NOs: 95 to 107,110, 116, 122, 129 to 131, 134 to 144, 149 to 172, and 175 to 179, inparticular SEQ ID NO: 134 corresponds to this illustration.

FIG. 1H. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1H shows an illustration of a recombinant binding proteinprovided herein comprising two designed ankyrin repeat domains withbinding specificity for serum albumin, and two designed ankyrin repeatdomains with binding specificities for other targets than serum albumin,linked by polypeptide linkers and having an N-terminal amino acidsequence. The two designed ankyrin repeat domains with bindingspecificity for serum albumin are N-terminal to the two other designedankyrin repeat domains. For example a recombinant binding protein withan amino acid sequence corresponding to any of SEQ ID NOs: 112, 119,124, 128, 132, and 133 corresponds to this illustration.

FIG. 1I. Illustration of recombinant binding proteins comprisingdesigned ankyrin repeat domains with binding specificity for serumalbumin. FIG. 1I. shows an illustration of a pharmaceutical compoundcomprising two designed ankyrin repeat domains with binding specificityfor serum albumin and a bioactive compound. The bioactive compound canbe covalently linked to the two designed ankyrin repeat domains withbinding specificity for serum albumin by means of chemical coupling or,in the case of polypeptides, protein fusion.

FIG. 2. Improved storage stability of recombinant binding proteinscomprising SEQ ID NO: 50. SDS 15% PAGE analysis of Proteins #49 and #50(corresponding to SEQ ID NOs: 49 and 50, respectively, additionallyhaving SEQ ID NO: 1 at the N terminus; prepared as described in Example4) stored at 10 mg/ml in PBS for 1 week at 4° C. (1), 25° C. (2), 40° C.(3), and 60° C. (4), respectively. M: Marker (lower band: 6.5 kDa; bandat Protein #50 level: 14.4 kDa; upper band in case of Protein #50 PAGE:21.5 kDa).

FIG. 3A. Mouse pharmacokinetic studies illustrating the benefit ofhaving two designed ankyrin repeat domains with binding specificity forserum albumin in a recombinant binding protein. Mouse pharmacokineticstudies were performed using ^(m99)Tc labeled proteins as described inExample 5. The percentage injected dose (% ID), referenced to an earlymeasurement time point (a: 4 h; b-d:1 h) is shown over time (t; hours).Proteins used comprised an N-terminal His-tag (SEQ ID NO: 1) in additionto the sequence indicated unless stated otherwise. FIG. 3A shows thepharmacokinetic profile comparison of Protein #57 (single designedankyrin repeat domain with binding specificity for serum albumin; SEQ IDNO: 57, which comprises SEQ ID NO: 51; filled circles) with Proteins #62and #63 (proteins comprising two designed ankyrin repeat domains withbinding specificity for serum albumin (twice SEQ ID NO: 51), linked byGS—(SEQ ID NO: 63; filled diamonds) or PT-rich (SEQ ID NO: 62; filledsquares) polypeptide linkers). Having two designed ankyrin repeatdomains with binding specificity for serum albumin leads to higher % IDat e.g. 24 h (+57% GS; +59% PT), 48 h (+76% GS; +82% PT) or 72 h (+79%GS; +94% PT) post-injection, and leads to an improved terminal half-life(+38% GS; +48% PT) compared to the protein comprising only a singledesigned ankyrin repeat domain with binding specificity for serumalbumin.

FIG. 3B. Mouse pharmacokinetic studies illustrating the benefit ofhaving two designed ankyrin repeat domains with binding specificity forserum albumin in a recombinant binding protein. Mouse pharmacokineticstudies were performed using m⁹⁹Tc labeled proteins as described inExample 5. The percentage injected dose (% ID), referenced to an earlymeasurement time point (a: 4 h; b-d:1 h) is shown over time (t; hours).Proteins used comprised an N-terminal His-tag (SEQ ID NO: 1) in additionto the sequence indicated unless stated otherwise. FIG. 3B shows thepharmacokinetic profile comparison of Protein #64 (filled circles),comprising SEQ ID NOs: 22 (designed ankyrin repeat domain with bindingspecificity for another target than serum albumin) and 51 (designedankyrin repeat domain with binding specificity for serum albumin), withProteins #73 (filled squares) and #74 (filled diamonds), comprising eachSEQ ID NOs: 22 and two times 51. Protein #73 has SEQ ID NOs: 51 flankingSEQ ID NO: 22, and Protein #74 has twice SEQ ID NOs: 51 N-terminal ofSEQ ID NO: 22. Having two designed ankyrin repeat domains with bindingspecificity for serum albumin leads to higher % ID at e.g. 24 h (+62%N-terminal; +89% flanking), or 48 h (+136% N-terminal; +175% flanking)post-injection, and leads to an improved terminal half-life (+>63% forboth N-terminal or flanking) compared to the protein comprising only asingle designed ankyrin repeat domain with binding specificity for serumalbumin.

FIG. 3C. Mouse pharmacokinetic studies illustrating the benefit ofhaving two designed ankyrin repeat domains with binding specificity forserum albumin in a recombinant binding protein. Mouse pharmacokineticstudies were performed using ^(m99)Tc labeled proteins as described inExample 5. The percentage injected dose (% ID), referenced to an earlymeasurement time point (a: 4 h; b-d:1 h) is shown over time (t; hours).Proteins used comprised an N-terminal His-tag (SEQ ID NO: 1) in additionto the sequence indicated unless stated otherwise. FIG. 3C shows thepharmacokinetic profile comparison of Protein #82 (filled circles),comprising SEQ ID NOs: 11 (twice; designed ankyrin repeat domain with noknown binding specificity) and 51 (designed ankyrin repeat domain withbinding specificity for serum albumin), with Protein #109 (filledsquares) comprising SEQ ID NOs: 11 (twice) and 51 (twice; N-terminal).Having two designed ankyrin repeat domains with binding specificity forserum albumin leads to higher % ID at e.g. 24 h (+12%), or 48 h (+35%)post-injection, and leads to an improved terminal half-life (+71%)compared to the protein comprising only a single designed ankyrin repeatdomain with binding specificity for serum albumin.

FIG. 3D. Mouse pharmacokinetic studies illustrating the benefit ofhaving two designed ankyrin repeat domains with binding specificity forserum albumin in a recombinant binding protein. Mouse pharmacokineticstudies were performed using ^(m99)Tc labeled proteins as described inExample 5. The percentage injected dose (% ID), referenced to an earlymeasurement time point (a: 4 h; b-d:1 h) is shown over time (t; hours).Proteins used comprised an N-terminal His-tag (SEQ ID NO: 1) in additionto the sequence indicated unless stated otherwise. FIG. 3D shows thepharmacokinetic profile comparison of Protein #83 (filled circles),comprising SEQ ID NOs: 38 and 39 (designed ankyrin repeat domains eachwith binding specificity for another target than serum albumin) and 50(designed ankyrin repeat domain with binding specificity for serumalbumin), with Proteins #110 (filled squares), comprising each SEQ IDNOs: 38, 39 and 50 (twice; flanking SEQ ID NOs: 38 and 39). Having twodesigned ankyrin repeat domains with binding specificity for serumalbumin leads to higher % ID at e.g. 24 h (+198%), 48 h (+198%), or 72 h(+228%) post-injection, and leads to an improved terminal half-life(+19%) compared to the protein comprising only a single designed ankyrinrepeat domain with binding specificity for serum albumin. Note that themeasurement of Protein #83 was close to the lower limit ofquantification.

FIG. 4A. Cynomolgus monkey pharmacokinetic studies illustrating thebenefit of having two designed ankyrin repeat domains with bindingspecificity for serum albumin in a recombinant binding protein.Cynomolgus monkey pharmacokinetic studies were performed as described inExample 6. The concentration of the respective protein is shown in nMover time indicated in days. Proteins used comprised an N-terminalHis-tag (SEQ ID NO: 1) in addition to the sequence indicated unlessstated otherwise. FIG. 4A shown the pharmacokinetic profile comparisonof Protein #57 (0.5 mg/Kg; 27.7 nmol/kg; single designed ankyrin repeatdomain with binding specificity for serum albumin; SEQ ID NO: 57, whichcomprises SEQ ID NO: 51; filled circles) with Protein #62 (1.04 mg/Kg;34.5 nmol/kg; protein comprising two designed ankyrin repeat domainswith binding specificity for serum albumin (twice SEQ ID NO: 51), linkedby a PT-rich polypeptide linker). Having two designed ankyrin repeatdomains with binding specificity for serum albumin leads to higherexposure (2138 d*nmol/L vs. 4676 d*nmol/L, i.e. +119% calculated up today 7), leads to a reduced clearance (0.0108 L/(d*kg) vs. 0.0031L/(d*kg); i.e. −71%), and leads to an improved terminal half-life (4.57d vs. 9.00 d, i.e. +97% calculated from day 1 to day 7) compared to theprotein comprising only a single designed ankyrin repeat domain withbinding specificity for serum albumin.

FIG. 4B. Cynomolgus monkey pharmacokinetic studies illustrating thebenefit of having two designed ankyrin repeat domains with bindingspecificity for serum albumin in a recombinant binding protein.Cynomolgus monkey pharmacokinetic studies were performed as described inExample 6. The concentration of the respective protein is shown asrelative value referenced to the measurement point at 10 minutespost-injection, over time indicated in hours. Proteins used comprised anN-terminal His-tag (SEQ ID NO: 1) in addition to the sequence indicatedunless stated otherwise. FIG. 4B shows the pharmacokinetic profile ofProtein #97 (a recombinant binding protein consisting of the amino acidsequence of SEQ ID NO: 97; filled squares) and Protein #134 (arecombinant binding protein consisting of the amino acid sequence of SEQID NO: 134, with no additional sequence tag; filled circles)administered at 1 mg/kg i.v. to cynomolgus monkeys are shown. Protein#134 has an improved pharmacokinetic profile compared to Protein #97.

FIG. 5. Size exclusion chromatography coupled to static light scatteringof a recombinant binding protein (Protein #134) comprising two designedankyrin repeat domains with binding specificity for serum albumin. Theexperiment was performed as described in Example 7 using Protein #134 (arecombinant binding protein consisting of SEQ ID NO: 134; solid line),human serum albumin (dotted line), and a mixture of the two (dashedline). The experiment indicates that Protein #134 comprising twodesigned ankyrin repeat domains (twice SEQ ID NO: 50) with bindingspecificity for serum albumin is able to bind two human serum albuminmolecules simultaneously.

FIG. 6A. Analysis of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134) inVEGF-A-binding ELISA, as outlined in Example 10. Protein #134 comprisesone designed ankyrin repeat domain with binding specificity for VEGF-A,one designed ankyrin repeat domain with binding specificity for HGF, andtwo designed ankyrin repeat domains with binding specificity for serumalbumin, and, correspondingly, interaction of Protein #134 with thesetarget proteins is expected. The binding signal of variousconcentrations of Protein #134 to immobilized VEGF-A of human (filledcircles), rat (filled squares), and mouse (filled rhombus), as well ashuman VEGF-C (open inverse triangles), and human PDGF-AB (open circles),and the corresponding fitting inhibition curves are shown. Protein #134binds VEGF-A of these species with high affinity and is not bindingVEGF-C and PDGF-AB.

FIG. 6B. Analysis of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134) in HGF-bindingELISA, as outlined in Example 10. Protein #134 comprises one designedankyrin repeat domain with binding specificity for VEGF-A, one designedankyrin repeat domain with binding specificity for HGF, and two designedankyrin repeat domains with binding specificity for serum albumin, and,correspondingly, interaction of Protein #134 with these target proteinsis expected. The binding signal of various concentrations of Protein#134 to immobilized HGF of human (filled circles), cynomolgus monkey(filled triangles), and mouse (filled rhombus), and the correspondingfitting inhibition curves are shown. Protein #134 binds HGF of thesespecies with high affinity.

FIG. 6C. Analysis of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134) inserum-albumin-binding ELISA, as outlined in Example 10. Protein #134comprises one designed ankyrin repeat domain with binding specificityfor VEGF-A, one designed ankyrin repeat domain with binding specificityfor HGF, and two designed ankyrin repeat domains with bindingspecificity for serum albumin, and, correspondingly, interaction ofProtein #134 with these target proteins is expected. The binding signalof various concentrations of Protein #134 to immobilized serum albuminof human (filled circles), cynomolgus monkey (open inverse triangle),rat (open triangle), mouse (open squares), and dog (open rhombus) andthe corresponding fitting inhibition curves are shown. Protein #134binds serum albumin of these species with high affinity. OD, opticaldensity at 450 nm minus OD at 620 nm; c [pM], concentration ofrecombinant binding protein in pM in logarithmic scale.

FIG. 7A. VEGF-A/VEGF-R2 and HGF/cMet receptor competition assays.Analysis of Protein #134 (a recombinant binding protein consisting ofthe amino acid sequence of SEQ ID NO: 134) in various competition assaysas described in Example 11. FIG. 7A shows the results of aVEGF-A/VEGFR-2 HTRF binding competition assay. Protein #134 inhibits theVEGF-A/VEGFR-2 interaction. Baseline is indicated by the dashed line, nocompetition signal is indicated by the circular symbol. R: ratio 665 nmsignal to 620 nm signal, c: concentration of Protein #134 in nM.

FIG. 7B. VEGF-A/VEGF-R2 and HGF/cMet receptor competition assays.Analysis of Protein #134 (a recombinant binding protein consisting ofthe amino acid sequence of SEQ ID NO: 134) in various competition assaysas described in Example 11. FIG. 7B shows the results of a HGF/cMetcompetition binding assay. Protein #134 inhibits the HGF/cMetinteraction. OD: OD at 450 nm minus OD at 620 nm, c: concentration ofProtein #134 in nM.

FIG. 7C. VEGF-A/VEGF-R2 and HGF/cMet receptor competition assays.Analysis of Protein #134 (a recombinant binding protein consisting ofthe amino acid sequence of SEQ ID NO: 134) in various competition assaysas described in Example 11. FIG. 7C shows the results of a VEGF-Acompetition binding ELISA. Protein #134 binds VEGF-A with an IC₅₀ ofbetter than 10 pM. OD: OD at 450 nm minus OD at 620 nm, c: concentrationof Protein #134 in pM.

FIG. 8. SPR analysis of recombinant binding proteins. Analysis of thebinding of VEGF-A, HGF, and HSA by Protein #134 (a recombinant bindingprotein consisting of the amino acid sequence of SEQ ID NO: 134) using aProtein instrument as described in Example 12. Human HGF is immobilizedon the biosensor chip, and Protein #134, human VEGF-A, or human serumalbumin were injected according to the following injection schemes: (1)Protein #134—hVEGF-A—HSA, (2) Protein #134—hVEGF-A—PBST, (3; dottedline) Protein #134—PBST—HSA, (4) PBST—PBST—PBST, (5) PBST—hVEGF-A—PBST,(6) PBST—PBST—HSA. Curves 1 and 2 indicate that Protein #134 can bindhuman HGF and human VEGF-A simultaneously. Furthermore, since VEGF-Abinding reaches saturation in curve 1, curve 1 indicates that Protein#134 can bind human HGF, human VEGF-A and human serum albuminsimultaneously. The control injections indicate that no unspecificinteraction occurs. RU: resonance units; t: time in seconds.

FIG. 9A. Effect of recombinant binding proteins cell proliferation andcell migration. The effect of Protein #134 (a recombinant bindingprotein consisting of the amino acid sequence of SEQ ID NO: 134) indifferent cellular assays was assessed as described in Example 13. FIG.9A shows inhibition of proliferation of HUVECs by Protein #134. HUVECs(3×10³ cells/well) were stimulated by 8 ng/mL human VEGF-A.Proliferative status of HUVECs was analyzed in the absence (open circle)or presence of increasing concentrations of Protein #134 (filledcircles). After 3 days of cultivation at 37° C. and 5% CO₂, inhibitionwas quantified by addition of BrdU for the last 24 h of incubation.Protein #134 exhibits an IC₅₀ in the range of 100-150 pM. Error barsreflect standard deviation of independent duplicates. OD: OD at 450 nmminus OD at 620 nm, c: concentration of Protein #134 in ng/ml. The Xaxis is shown in logarithmic scale.

FIG. 9B. Effect of recombinant binding proteins cell proliferation andcell migration. The effect of Protein #134 (a recombinant bindingprotein consisting of the amino acid sequence of SEQ ID NO: 134) indifferent cellular assays was assessed as described in Example 13. FIG.9B shows the effect of Protein #134 in an Oris cell migration assay withA549 cells. Protein #134 significantly inhibits the HGF-induced cellmigration. Cells were seeded 24 h prior to stimulation with HGF (500 μM;H and D) or PBS (N), in the presence (D) and absence (H, N) of Protein#134 (5 μM). The stoppers were removed and migration was detected andquantified 48 h later after staining of cells with Calcein. Images weretaken and the uncovered area in the cell culture plate well quantified.U: Uncovered area in 8 independent wells in μm², H: HGF, no Protein#134, N: No HGF, no Protein #134, D: HGF & Protein #134.

FIG. 9C. Effect of recombinant binding proteins cell proliferation andcell migration. The effect of Protein #134 (a recombinant bindingprotein consisting of the amino acid sequence of SEQ ID NO: 134) indifferent cellular assays was assessed as described in Example 13. FIG.9C shows inhibition of cMet phosphorylation in A549 cells by Protein#134. A549 cells were starved overnight and stimulated with 1 nM humanHGF (no HGF for negative control) in presence of PBS or increasingconcentrations of Protein #134 for 10 minutes. P-cMet was detected incell lysates by ELISA measuring OD450-620. Relative signals (%phosphorylation) were calculated using maximal signal (HGF, no Protein#134) and minimal signal (no HGF, no Protein #134). Protein #134inhibits cMet phosphorylation with an IC₅₀ of better than 1 nM. % P: %phosphorylation, c: concentration of Protein #134 in nM.

FIG. 10A. Effect of recombinant binding proteins on tumor growth invivo. The efficacy of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134; comprises (i)one designed ankyrin repeat domain with binding specificity for VEGF-A,(ii) one designed ankyrin repeat domain with binding specificity forHGF, and (iii) two designed ankyrin repeat domains with bindingspecificity for serum albumin; see FIGS. 1A-1I) and of other recombinantbinding proteins was assessed in tumor xenograft mouse models asdescribed in Example 14. FIG. 10A shows the quantification ofproliferative cells and mean vascular area in tumor tissue of a U87Mmouse model treated with Protein #134, Protein #60 (a recombinantbinding protein consisting of the amino acid sequence of SEQ ID NO: 60and additionally having SEQ ID NO: 1 at the N-terminus; comprises onedesigned ankyrin repeat domain with binding specificity for HGF(identical to the one in Protein #134), and one designed ankyrin repeatdomain with binding specificity for serum albumin (identical to the onein Protein #134)), or Protein #61 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 61 and additionallyhaving SEQ ID NO: 1 at the N-terminus; comprises one designed ankyrinrepeat domain with binding specificity for VEGF-A (identical to the onein Protein #134), and one designed ankyrin repeat domain with bindingspecificity for serum albumin (identical to the one in Protein #134)),as described in Example 14. Regarding inhibition of the proliferation ofU87M tumor xenograft cells (P; measured as percent proliferative cells,% pc), Protein #60 exhibits a slight inhibition, similar as Protein #61,whereas Protein #134 has a significantly stronger effect. Likewise,regarding inhibition of vascular growth (A; measured as mean vasculararea percentage; % mva), Protein #60 exhibits a slight inhibition,Protein #61 exhibits an intermediate inhibition, and Protein #134exhibits the strongest effect. PBS (white bars), Protein #60(horizontally striped bars), Protein #61 (vertically striped bars),Protein #134 (black bars).

FIG. 10B. Effect of recombinant binding proteins on tumor growth invivo. The efficacy of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134; comprises (i)one designed ankyrin repeat domain with binding specificity for VEGF-A,(ii) one designed ankyrin repeat domain with binding specificity forHGF, and (iii) two designed ankyrin repeat domains with bindingspecificity for serum albumin; see FIGS. 1A-1I) and of other recombinantbinding proteins was assessed in tumor xenograft mouse models asdescribed in Example 14. FIG. 10B shows the effect of Protein #134 ontumor growth in a patient derived renal tumor xenograft mouse model incomparison to sorafenib and PBS. Details of the model are described inExample 14. Sorafenib suppresses tumor growth as expected.Interestingly, Protein #134 suppresses tumor growth beyond the levels ofsorafenib, controlling the tumor volume at its initial levels. V: tumorvolume in mm³, d: days of treatment, open circles: vehicle, closedcircles: Protein #134 (4 mg/kg), open square: sorafenib (200 mg/kg).

FIG. 10C. Effect of recombinant binding proteins on tumor growth invivo. The efficacy of Protein #134 (a recombinant binding proteinconsisting of the amino acid sequence of SEQ ID NO: 134; comprises (i)one designed ankyrin repeat domain with binding specificity for VEGF-A,(ii) one designed ankyrin repeat domain with binding specificity forHGF, and (iii) two designed ankyrin repeat domains with bindingspecificity for serum albumin; see FIGS. 1A-1I) and of other recombinantbinding proteins was assessed in tumor xenograft mouse models asdescribed in Example 14. FIG. 10C shows the effect of Protein #134 ontumor growth in a patient derived gastric tumor xenograft mouse model incomparison to paclitaxel and a combination of Protein #134 andpaclitaxel. Details of the model are described in Example 14. Paclitaxeland Protein #134 suppress tumor growth to about the same level. Thecombination of Paclitaxel and Protein #134 suppresses tumor growth evenmore, controlling the tumor volume at its initial levels. V: tumorvolume in mm³, d: days post therapy, open circles: vehicle, closedcircles: Protein #134 (4 mg/kg i.v.), open square: paclitaxel (15 mg/kgi.v.), open triangles: Protein #134 (4 mg/kg) and paclitaxel (15 mg/kg).

DETAILED DESCRIPTION

In the context of the present invention the term “protein” refers to apolypeptide, wherein at least part of the polypeptide has, or is able toacquire a defined three-dimensional arrangement by forming secondary,tertiary, or quaternary structures within a single polypeptide chainand/or between multiple polypeptide chains. If a protein comprises twoor more polypeptide chains, the individual polypeptide chains may belinked non-covalently or covalently, e.g. by a disulfide bond betweentwo polypeptides. A part of a protein, which individually has, or isable to acquire, a defined three-dimensional arrangement by formingsecondary or tertiary structure, is termed “protein domain”. Suchprotein domains are well known to the practitioner skilled in the art.

The term “recombinant” as used in recombinant protein, recombinantprotein domain, recombinant binding protein and the like, means thatsaid polypeptides are produced by the use of recombinant DNAtechnologies well known by the practitioner skilled in the relevant art.For example, a recombinant DNA molecule (e.g. produced by genesynthesis) encoding a polypeptide can be cloned into a bacterialexpression plasmid (e.g. pQE30, QIAgen), yeast expression plasmid,mammalian expression plasmid, or plant expression plasmid, or a DNAenabling in vitro expression. If, for example, such a recombinantbacterial expression plasmid is inserted into an appropriate bacteria(e.g. Escherichia coli), this bacteria can produce the polypeptideencoded by this recombinant DNA. The correspondingly producedpolypeptide is called a recombinant polypeptide or recombinant protein.

In the context of the present invention, the term “binding protein”refers to a protein comprising two or more, preferably three or more,more preferably four or more binding domains. Preferably, said bindingprotein is a recombinant binding protein. Preferably, said bindingprotein comprises two or more repeat domains. More preferably, saidbinding protein comprises three repeat domains. More preferably, saidbinding protein comprises four repeat domains. Also preferably, saidbinding protein comprises three or more designed ankyrin repeat domains.Further preferably, said binding protein comprises four or more designedankyrin repeat domains. More preferably, said binding protein comprisesfour designed ankyrin repeat domains. Optionally, said binding proteincomprises one or more bioactive compound. Said binding domains of saidbinding protein each have a target specificity. Preferably, two or moreof said binding domains of said binding protein each have a targetspecificity for serum albumin. Preferably, said binding proteincomprises at least three binding domains binding to at least twodifferent targets. More preferably, said binding protein comprises atleast four binding domains binding to at least three different targets.

Furthermore, any such binding protein may comprise additionalpolypeptides (such as e.g. polypeptide tags, or polypeptide linkers,well known to the person skilled in the art.

The term “bioactive compound” refers to a compound that is diseasemodifying when applied to a mammal having said disease. A bioactivecompound may have antagonistic or agonistic properties and can be aproteinaceous bioactive compound or a non-proteinaceous bioactivecompound. Such proteinaceous bioactive compounds can be covalentlyattached to, for example, a binding domain of the invention by thegeneration of genetic fusion polypeptides using standard DNA cloningtechnologies, followed by their standard expression and purification.Non-proteinaceous bioactive compounds can be covalently attached to, forexample, a binding domain of the invention by chemical means, e.g., bycoupling to a cysteine thiol via a maleimide linker with a cysteinebeing coupled via a polypeptide linker to the N or C terminus of abinding domain as described hereinbefore. Examples of proteinaceousbioactive compounds are binding domains having a distinct targetspecificity (e.g. neutralizing a growth factor by binding to it),cytokines (e.g. interleukins), growth factors (e.g. human growthhormone), antibodies and fragments thereof, hormones (e.g. GLP-1), or aproteinaceous drug. Examples of non-proteinaceous bioactive compoundsare toxins (e.g. DM1 from ImmunoGen), small molecules targeting GPCRs,antibiotics or a non-proteinaceous drug.

The term “binding domain” means a protein domain exhibiting the same“fold” (i.e. secondary, tertiary, and/or quaternary structure) as aprotein scaffold and having a predetermined property, as defined below.Such a binding domain may be obtained by rational, or most commonly,combinatorial protein engineering techniques, skills which are known inthe art (Binz et al., 2005, loc. cit.). For example, a binding domainhaving a predetermined property can be obtained by a method comprisingthe steps of (a) providing a diverse collection of protein domainsexhibiting the same fold as a protein scaffold as defined further below;and (b) screening said diverse collection and/or selecting from saiddiverse collection to obtain at least one protein domain having saidpredetermined property. The diverse collection of protein domains may beprovided by several methods in accordance with the screening and/orselection system being used, and may comprise the use of methods wellknown to the person skilled in the art, such as phage display orribosome display. Preferably, said binding domain is a recombinantbinding domain.

The term “protein scaffold” means a protein with exposed surface areasin which amino acid insertions, substitutions or deletions are highlytolerable. Examples of protein scaffolds that can be used to generatebinding domains of the present invention are antibodies or fragmentsthereof such as single-chain Fv or Fab fragments, protein A fromStaphylococcus aureus, the bilin binding protein from Pieris brassicaeor other lipocalins, ankyrin repeat proteins or other repeat proteins,and human fibronectin. Protein scaffolds are known to the person skilledin the art (Binz et al., 2005, loc. cit.; Binz et al., 2004, loc. cit.).

The term “target” refers to an individual molecule such as a nucleicacid molecule, a polypeptide or protein, a carbohydrate, or any othernaturally occurring molecule, including any part of such individualmolecule, or complexes of two or more of such molecules. A target may bea whole cell or a tissue sample, or it may be any non-natural compound.Preferably, a target is a naturally occurring or non-natural polypeptideor a polypeptide containing chemical modifications, for example modifiedby natural or non-natural phosphorylation, acetylation, or methylation.In the particular application of the present invention, the targets areserum albumin, HGF and VEGF-A.

The term “predetermined property” refers to a property such as bindingto a target, blocking of a target, activation of a target-mediatedreaction, enzymatic activity, and related further properties. Dependingon the type of desired property, one of ordinary skill will be able toidentify format and necessary steps for performing screening and/orselection of a binding domain with the desired property. Preferably,said predetermined property is specifically binding to a target.

In the context of the present invention, the term “polypeptide” relatesto a molecule consisting of a chain of multiple, i.e. two or more, aminoacids linked via peptide bonds.

Preferably, a polypeptide consists of more than eight amino acids linkedvia peptide bonds. The term “polypeptide” also includes multiple chainsof amino acids, linked together by S—S bridges of cysteines.Polypeptides are well-known to the person skilled in the art.

The term “polypeptide tag” refers to an amino acid sequence attached toa polypeptide/protein, wherein said amino acid sequence is useful forthe purification, detection, or “targeting” (i.e. localization to thesite of a target) of said polypeptide/protein, or wherein said aminoacid sequence improves the physicochemical behavior of thepolypeptide/protein, or wherein said amino acid sequence possesses aneffector function. The individual polypeptide tags of a binding proteinmay be connected to other parts of the binding protein directly or viapolypeptide linkers. These polypeptide tags are all well known in theart and are fully available to the person skilled in the art. Examplesof polypeptide tags are small polypeptide sequences, for example, His(e.g. the His-tag of SEQ ID NO: 1), myc, FLAG, or Strep-tags, orpolypeptides such as enzymes (for example alkaline phosphatase), whichallow the detection of said polypeptide/protein, or polypeptides whichcan be used for targeting (such as immunoglobulins or fragments thereof)and/or as effector molecules.

The term “polypeptide linker” refers to an amino acid sequence, which isable to link, for example, two protein domains, a polypeptide tag and aprotein domain, a protein domain and a non-proteinaceous compound orpolymer such as polyethylene glycol, or two sequence tags. Suchadditional domains, tags, non-proteinaceous compounds or polymers andlinkers are known to the person skilled in the relevant art. A list ofexamples is provided in the description of patent application WO2002/020565. Particular examples of such linkers areglycine-serine-linkers and proline-threonine-linkers of variablelengths; preferably, said linkers have a length between 2 and 30 aminoacids; more preferably, said linkers have a length between 2 and 24amino acids. Examples of glycine-serine-linkers are GS and amino acidsequences provided in SEQ ID NOs: 2 to 6, and examples ofproline-threonine-linkers are provided in amino acid sequences SEQ IDNOs: 7 to 9.

Patent application WO 2002/020565 and Forrer et al., 2003 (loc. cit.),contain a general description of repeat protein features and repeatdomain features, techniques and applications. The term “repeat protein”refers to a protein comprising one or more repeat domains. Preferably, arepeat protein comprises up to six repeat domains. More preferably, arepeat protein comprises up to five repeat domains. More preferably, arepeat protein comprises up to four repeat domains. Furthermore, saidrepeat protein may comprise additional non-repeat protein domains,polypeptide tags and/or polypeptide linkers. The repeat domains can bebinding domains as described hereinbefore.

The term “repeat domain” refers to a protein domain comprising two ormore consecutive repeat modules as structural units, wherein saidstructural units have the same fold, and stack tightly to create asuperhelical structure having a joint hydrophobic core. Next to astructural homology, such repeat modules further have a sequencehomology. Preferably, a repeat domain further comprises an N-terminaland/or a C-terminal capping repeat. For clarity, a capping repeat can bea repeat module. Such repeat domains, repeat modules, and cappingrepeats, sequence motives, as well as structural homology and sequencehomology are well known to the practitioner in the art from examples ofdesigned ankyrin repeat domains (WO 2002/020565), leucine-rich repeatdomains (WO 2002/020565), tetratricopeptide repeat domains (Main, E. R.,Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Structure 11(5),497-508, 2003), and armadillo repeat domains (WO 2009/040338). It isfurther well known to the practitioner in the art, that such repeatdomains are different from proteins comprising repeated amino acidsequences, where every repeated amino acid sequence is able to form anindividual domain (for example FN3 domains of Fibronectin), or where therepeated amino acid sequences are no structural units, i.e. saidrepeated amino acid sequences do not stack tightly to create asuperhelical structure having a joint hydrophobic core. Methods foridentifying and determining repeat modules or repeat sequence motifs orfor identifying families of related proteins comprising such repeatunits or motifs, such as homology searches (BLAST etc.), are wellestablished in the field of bioinformatics, and are well known to thepractitioner in the art.

The term “designed repeat protein” and “designed repeat domain” refer toa repeat protein or repeat domain, respectively, obtained as the resultof an inventive procedure, e.g. as explained in patent application WO2002/020565. The term “designed” refers to the property that such repeatproteins and repeat domains, respectively, are man-made, synthetic andnot from nature. The designed repeat proteins or designed repeat domainsof WO 2002/020565 include designed ankyrin repeat proteins or designedankyrin repeat domains, respectively. Accordingly, a designed ankyrinrepeat protein herein corresponds to protein of the invention comprisingat least one designed ankyrin repeat domain. Further, the term “not fromnature” means that the sequence of said binding protein or said bindingdomain is not present as a non-artificial sequence entry in a sequencedatabase, for example in GenBank, EMBL-Bank or Swiss-Prot. Thesedatabases and other similar sequence databases are well known to theperson skilled in the art. The recombinant binding proteins or designedankyrin repeat domains of the invention are non-naturally occurring.

The terms “repeat module”, “repeat unit”, “capping repeat”, “cappingmodule”, and further terms relating to repeat proteins and repeatdomains, are defined in WO 2002/020565, and the definitions areincorporated by reference.

The term “has binding specificity for a target”, “specifically bindingto a target”, “binding to a target with high specificity”, “specific fora target” or “target specificity” and the like means that a bindingprotein or binding domain binds in PBS to a target with a lowerdissociation constant (i.e. it binds with higher affinity) than it bindsto an unrelated protein such as the E. coli maltose binding protein(MBP). Preferably, the dissociation constant (“Kd”) in PBS for thetarget is at least 10²; more preferably, at least 10³; more preferably,at least 10⁴; or more preferably, at least 10⁵ times lower than thecorresponding dissociation constant for MBP.

Methods to determine dissociation constants of protein-proteininteractions, such as surface plasmon resonance (SPR) based technologies(e.g. SPR equilibrium analysis) or isothermal titration calorimetry(ITC) are well known to the person skilled in the art. The measured Kdvalues of a particular protein-protein interaction can vary if measuredunder different conditions (e.g., salt concentration, pH). Thus,measurements of Kd values are preferably made with standardizedsolutions of protein and a standardized buffer, such as PBS.

The term “PBS” means a phosphate buffered water solution containing 137mM NaCl, 10 mM phosphate and 2.7 mM KCl and having a pH of 7.4.

The term “inhibits the binding” in the context of the binding domains ofthe present invention refers to the ability of said binding domains toprevent the binding of its target to another protein, typically anatural ligand of the target or another antagonist. The strength ofinhibition is typically measured by assessing the concentration ofhalf-maximal inhibition (IC₅₀). The term inhibition and the assessmentof IC₅₀ values are well established in the field. For example, thedesigned ankyrin repeat domain of SEQ ID NO: 18 inhibits the binding ofVEGF-A to its natural ligand VEGFR-2.

The invention relates to designed ankyrin repeat domains with bindingspecificity for serum albumin, and to recombinant binding proteinscomprising at least two designed ankyrin repeat domains with bindingspecificity for serum albumin, and to recombinant binding proteinscomprising at least a first, a second, a third, and a fourth designedankyrin repeat domain, wherein said first designed ankyrin repeat domainhas binding specificity for VEGF-A, and wherein said second designedankyrin repeat domain has binding specificity for HGF, and wherein saidthird and fourth designed ankyrin repeat domains each have bindingspecificity for serum albumin.

In one embodiment, the invention relates to designed ankyrin repeatdomains with binding specificity for serum albumin. Examples of designedankyrin repeat domains with binding specificity for serum albumin aregiven in SEQ ID NOs: 40 to 56 (see also Examples) and further examplesare described in WO 2012/069654. In particular, the invention relates todesigned ankyrin repeat domains with binding specificity for serumalbumin selected from the group of SEQ ID NOs: 48 to 50, more preferablySEQ ID NOs: 49 and 50, more preferably SEQ ID NO: 50, in which up to 10,9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acids are exchanged by any aminoacid. In one embodiment, the invention relates to designed ankyrinrepeat domains with binding specificity for serum albumin that have 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith a designed ankyrin repeat domain selected from the group of SEQ IDNOs: 48 to 50, more preferably SEQ ID NOs: 49 and 50, more preferablySEQ ID NO: 50. In one embodiment, the invention relates to designedankyrin repeat domains with binding specificity for serum comprising anamino acid sequence selected from the group of SEQ ID NOs: 48 to 50,more preferably SEQ ID NOs: 49 and 50, more preferably SEQ ID NO: 50. Inone embodiment, the invention relates to designed ankyrin repeat domainswith binding specificity for serum selected that consist of an aminoacid sequence selected from the group of SEQ ID NOs: 48 to 50, morepreferably SEQ ID NOs: 49 and 50, more preferably SEQ ID NO: 50. In oneembodiment, the invention relates to a designed ankyrin repeat domainwith binding specificity for serum albumin comprising the amino acidsequence of SEQ ID NO: 50. The preferred designed ankyrin repeat domainwith binding specificity for serum albumin of the invention is SEQ IDNO: 50. Preferably, said designed ankyrin repeat domain with bindingspecificity for serum albumin binds serum albumin of mouse, rat, dog,cynomolgus monkey, or human origin, more preferably serum albumin ofmouse, cynomolgus monkey or human origin, more preferably serum albuminof cynomolgus monkey or human origin, more preferably serum albumin ofhuman origin, in PBS with a dissociation constant (Kd) below 10⁻⁵M;preferably below 10⁻⁶M; or more preferably below 10⁻⁷M. The term “mouseserum albumin” refers to UniProt accession number P07724, the term“cynomolgus monkey serum albumin” (i.e. macaca fascicularis) refers toUniProt accession number A2V9Z4, and the term “human serum albumin”refers to UniProt accession number P02768. In one embodiment, theinvention relates to a designed ankyrin repeat domain with bindingspecificity for serum albumin comprising, more preferably consisting of,an amino acid sequence selected from the group of SEQ ID NOs: 48 to 50,more preferably SEQ ID NOs: 49 and 50, more preferably SEQ ID NO: 50,which exhibit improved storage stabilities compared to SEQ ID NO: 51.“Improved storage stability” in the context of the present inventionmeans an improved midpoint of denaturation temperature (i.e. midpoint ofthe cooperative unfolding upon temperature increase) by 0.5° C., 1° C.,1.5° C., 2° C., 2.5° C., 3° C., 3.5° C., or 4° C., and/or the reductionof the amounts of a degradation band, preferably the reduction of theamount of degradation products, as detected by a Coomassie-stainedSDS-PAGE occurring after storage at 40° C. for 1 month at 10 mg/ml inPBS, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.Methods to assess storage stability by SDS-PAGE and methods to determinethe midpoint of denaturation by using fluorimetric methods or circulardichroism are well known to the person skilled in the art. In oneembodiment, the invention relates to a designed ankyrin repeat domainwith binding specificity for serum albumin comprising, more preferablyconsisting of amino acid sequence SEQ ID NO: 50, which exhibits improvedstorage stability compared to SEQ ID NO: 49, preferably which exhibitsreduced amounts of degradation products, as detected by SDS-PAGE,occurring after storage at 40° C. for 1 month at 10 mg/ml in PBS, by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, compared toSEQ ID NO: 49. Examples of designed ankyrin repeat domains andrecombinant binding proteins with improved storage stability propertiesare given in Example 9.

In one embodiment, the invention relates to a designed ankyrin repeatdomain with binding specificity for serum albumin selected from thegroup consisting of amino acid sequences SEQ ID NOs: 44 to 49, 51 and52, more preferably 48, 49, 51, and 52, more preferably 48 and 49, morepreferably 49, comprising Glutamate at position 78. In one embodiment,the invention relates to SEQ ID NO: 49 wherein Aspartate at position 78has been exchanged by Glutamate, corresponding to SEQ ID NO: 50. SEQ IDNO: 49 comprises a high number of potential degradation sites.Degradation may for example occur in the vicinity of any one of the 5asparagines (including asparagine-glycine dipeptides), 13 aspartates, or10 glycines of SEQ ID NO: 49, amongst additional potential degradationsites. SEQ ID NO: 49 further comprises a number of potential oxidationsites. Surprisingly, a major effect on storage stability can be achievedby mutating only position 78 of SEQ ID NO: 49. Furthermore, thefunctionality of the designed ankyrin repeat with binding specificityfor serum albumin can be preserved by mutating position 78 of SEQ ID NO:49 from aspartate to glutamine. The designed ankyrin repeat domainconsisting of SEQ ID NO: 49 comprising Glutamate in position 78 exhibitshigher storage stability compared to the designed ankyrin repeat domaincomprising Aspartate in that position.

In one embodiment, the present invention relates to a recombinantbinding protein comprising at least two, preferably comprising two,designed ankyrin repeat domains with binding specificity for serumalbumin. The preferred recombinant binding protein of the inventioncomprises two designed ankyrin repeat domains with binding specificityfor serum albumin. Examples of such recombinant binding proteins aregiven in the amino acid sequences SEQ ID NOs: 62, 63, 73 to 81, and 95to 179.

In one embodiment, the present invention relates to a recombinantbinding protein comprising at least two, more preferably comprising two,designed ankyrin repeat domains with binding specificity for serumalbumin, wherein said recombinant binding protein exhibits improvedpharmacokinetic properties compared to the recombinant binding proteincomprising only one designed ankyrin repeat domain with bindingspecificity for serum albumin. The examples of the present inventiondisclose such recombinant binding proteins.

The expression “the recombinant binding protein comprising only onedesigned ankyrin repeat domain with binding specificity for serumalbumin”, means a recombinant binding that has the composition of arecombinant binding protein of the present invention in which the numberof designed ankyrin repeat domains with binding specificity for serumalbumin is reduced to one, by removing all designed ankyrin repeatdomains with binding specificity for serum albumin but one, and thecorresponding polypeptide linkers. Preferably, said remaining onedesigned ankyrin repeat domain with binding specificity for serumalbumin is located at a position in the recombinant binding proteincorresponding to a position that was comprising a designed ankyrinrepeat domain with binding specificity for serum albumin in therecombinant binding protein of the present invention, and the remainingone designed ankyrin repeat domain with binding specificity for serumalbumin is identical to the designed ankyrin repeat domain with bindingspecificity for serum albumin that was at the corresponding position inthe recombinant binding protein of the present invention. For example,the recombinant binding protein consisting of SEQ ID NO: 85 is therecombinant binding protein consisting of SEQ ID NO: 95, in which theC-terminal designed ankyrin repeat domain with binding specificity forserum albumin (in this case SEQ ID NO: 50) as well as the adjacentpolypeptide linker (in this case SEQ ID NO: 9) have been removed.Importantly, the remaining designed ankyrin repeat domain with bindingspecificity for serum albumin (SEQ ID NO: 50) of SEQ ID NO: 85 isN-terminal, and SEQ ID NO: 95 comprises the same SEQ ID NO: 50 at thesame position. Likewise, the recombinant binding protein consisting ofSEQ ID NO: 83 is the recombinant binding protein consisting of SEQ IDNO: 110, in which the C-terminal designed ankyrin repeat domain withbinding specificity for serum albumin (in this case SEQ ID NO: 50) aswell as the adjacent polypeptide linker (in this case SEQ ID NO: 9) havebeen removed.

The expression “exhibits improved pharmacokinetic properties”, “improvedpharmacokinetic properties”, or “pharmacokinetic property improvement”in this invention has the meaning that a pharmacokinetic parameter of arecombinant binding protein is improved compared to the correspondingpharmacokinetic parameter of a protein it is compared with.Corresponding examples are shown in Examples 5 and 6 and FIGS. 3A-3D andFIGS. 4A-4B. For example, when comparing Protein #110 (a proteinconsisting of SEQ ID NO: 110 and additionally SEQ ID NO: 1 at the Nterminus) with Protein #83 (a protein consisting of SEQ ID NO: 83 andadditionally SEQ ID NO: 1 at the N terminus) in cynomolgus monkeypharmacokinetic studies Protein #110 has a higher exposure (+32%), areduced clearance (−47%) as well as a higher terminal half-life (+168%,calculated from day 1 to day 6) as Protein #83. As another example, whencomparing Protein #62 (a protein consisting of SEQ ID NO: 62 andadditionally SEQ ID NO: 1 at the N terminus) with Protein #57 (a proteinconsisting of SEQ ID NO: 57 and additionally SEQ ID NO: 1 at the Nterminus) in cynomolgus monkey pharmacokinetic studies Protein #62 has ahigher exposure (+119%), a reduced clearance (−71%) as well as a higherterminal half-life (+97%, calculated from day 1 to day 7) as Protein#57. Or when comparing Protein #109 (a protein consisting of SEQ ID NO:109 and additionally SEQ ID NO: 1 at the N terminus) with Protein #82 (aprotein consisting of SEQ ID NO: 82 and additionally SEQ ID NO: 1 at theN terminus) in cynomolgus monkey pharmacokinetic studies Protein #109has a higher exposure (+19%), a reduced clearance (−37%) as well as ahigher terminal half-life (+55%, calculated from day 1 to day 7) asProtein #82. As yet another example, when comparing Protein #97 (aprotein consisting of SEQ ID NO: 97 and additionally SEQ ID NO: 1 at theN terminus) with Protein #68 (a protein consisting of SEQ ID NO: 68 andadditionally SEQ ID NO: 1 at the N terminus) in cynomolgus monkeypharmacokinetic studies Protein #97 has a higher terminal half-life(+264%, calculated from day 1 to day 7) as Protein #68. Further examplesare given in Examples 5 and 6 as well as FIGS. 3A-3D and FIGS. 4A-4B.Preferably, an improved pharmacokinetic property is a reduced clearance,and/or an increased exposure, and/or an increased terminal half-life.More preferably, an improved pharmacokinetic property is an increasedterminal half-life. In one embodiment, a recombinant binding protein ofthe present invention, comprising at least two, more preferablycomprising two, designed ankyrin repeat domains with binding specificityfor serum albumin exhibits an increased terminal half-life, and/or areduced clearance, and/or an increased exposure of at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,200%, or 250% compared to the recombinant binding protein comprisingonly one designed ankyrin repeat domain with binding specificity forserum albumin. In one embodiment, a recombinant binding protein of thepresent invention, comprising at least two, more preferably comprisingtwo, designed ankyrin repeat domains with binding specificity for serumalbumin exhibits an increased terminal half-life, preferably anincreased terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%compared to the recombinant binding protein comprising only one designedankyrin repeat domain with binding specificity for serum albumin.

Preferably, clearance, and/or exposure, and/or terminal half-life areassessed in a mammal, more preferably mouse and/or cynomolgus monkey,more preferably cynomolgus monkey. Preferably, when measuring theclearance, and/or exposure, and/or terminal half-life in mouse, theevaluation is done considering the data up to 48 h post-injection. Morepreferably, the evaluation of terminal half-life in mouse is calculatedfrom 24 h to 48 h. Preferably, when measuring the clearance, and/orexposure, and/or terminal half-life in cynomolgus monkey, the evaluationis done considering the data up to day 7 post-injection. Morepreferably, the evaluation of terminal half-life in cynomolgus monkey iscalculated from day 1 to day 7. The term “terminal half-life” of a drugsuch as a recombinant binding protein of the invention refers to thetime required to reach half the plasma concentration of the drug appliedto a mammal after reaching pseudo-equilibrium (for example calculatedfrom 24 h to 48 h in mouse or calculated from day 1 to day 7 incynomolgus monkey). Terminal half-life is not defined as the timerequired to eliminate half the dose of the drug administered to themammal. The term terminal half-life is well known to the person skilledin the art. Preferably, pharmacokinetic comparison is done at any dose,more preferably at equivalent dose (i.e. same mg/kg dose) or equimolardose (i.e. same mol/kg dose), more preferably at equimolar dose (i.e.same mol/kg dose). It is understood by the person skilled in the artthat equivalent and/or equimolar dosing in animals is subject toexperimental dose variations of at least 20%, more preferably 30%, 40%,50%, 60%, 70%, 80%, 90%, or 100%. Preferably, a dose used forpharmacokinetic measurement is selected from 0.001 to 1000 mg/kg, morepreferably 0.01 to 100 mg/kg, more preferably 0.1 to 50 mg/kg, morepreferably 0.5 to 10 mg/kg.

In one embodiment, a recombinant binding protein of the presentinvention comprising at least two, more preferably comprising two,designed ankyrin repeat domains with binding specificity for serumalbumin, exhibits a higher percentage of injected dose in mouse 24 hand/or 48 h and/or 72 h post injection, preferably 24 h post-injection,preferably 48 h post-injection, more preferably 72 h post-injection,more preferably 72 h and 48 h post-injection, more preferably 24 h, 48 hand 72 h post-injection, compared to the recombinant binding proteincomprising only one designed ankyrin repeat domain with bindingspecificity for serum albumin. Preferably, the percentage of injecteddose in mouse is calculated by comparison to the concentrationmeasurement 1 h or 4 h, preferably 1 post-injection. In one embodiment,the recombinant binding protein of the present invention comprising atleast two, more preferably comprising two, designed ankyrin repeatdomains with binding specificity for serum albumin, exhibits a higherpercentage of injected dose in cynomolgus monkey 4 days and/or 5 daysand/or 6 days post-injection, preferably 4 days, preferably 5 days, morepreferably 6 days, more preferably 5 and 6 days post-injection, morepreferably 4, 5, and 6 days post-injection, compared to the recombinantbinding protein comprising only one designed ankyrin repeat domain withbinding specificity for serum albumin. Preferably, the percentage ofinjected dose in cynomolgus monkey is calculated by comparison to theconcentration measurement 10 min or 1 h, preferably 10 minpost-injection. A higher percentage of injected dose refers to anincreased percentage of dose of at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%.

In one embodiment, the recombinant binding protein of the presentinvention comprises at least three designed ankyrin repeat domains,wherein at least two designed ankyrin repeat domains are designedankyrin repeat domains with binding specificity for serum albumin.Examples of such recombinant binding proteins are given in the aminoacid sequences SEQ ID NOs: 73 to 81 and 95 to 179.

In one embodiment, the recombinant binding protein comprises at leastfour designed ankyrin repeat domains, wherein at least two designedankyrin repeat domains are designed ankyrin repeat domains with bindingspecificity for serum albumin. Examples of such recombinant bindingproteins are given in the amino acid sequences SEQ ID NOs: 95 to 179.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein each of said designedankyrin repeat domains with binding specificity for serum albumin in PBShas binding specificity for serum albumin of mammalian origin, morepreferably mouse, rat, dog, cynomolgus monkey, or human origin, morepreferably serum albumin of mouse, cynomolgus monkey or human origin,more preferably serum albumin of cynomolgus monkey or human origin, morepreferably serum albumin of human origin.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein each of said designedankyrin repeat domains with binding specificity for serum albumin bindsserum albumin, more preferably serum albumin of mammalian origin, morepreferably serum albumin of mouse, rat, dog, cynomolgus monkey, or humanorigin, more preferably serum albumin of mouse, cynomolgus monkey orhuman origin, more preferably serum albumin of cynomolgus monkey orhuman origin, preferably serum albumin of human origin, in PBS with adissociation constant (Kd) below 10⁻⁵M, preferably below 10⁻⁶M, morepreferably below 10⁻⁷M. Examples of such designed ankyrin repeat domainswith binding specificity for serum albumin are given in Example 2 and inSEQ ID NOs: 40 to 56.

In one embodiment, the present invention relates to a recombinantbinding protein comprising two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said two designed ankyrinrepeat domains with binding specificity for serum albumin are at anyposition compared to any other protein domain, preferably any otherdesigned ankyrin repeat domain, comprised in said recombinant bindingprotein, preferably wherein said two designed ankyrin repeat domainswith binding specificity for serum albumin are both N-terminal of anyother protein domain, preferably any other designed ankyrin repeatdomain, comprised in said recombinant binding protein, or wherein saidtwo designed ankyrin repeat domains with binding specificity for serumalbumin are one N-terminal and one C-terminal of any other proteindomain, preferably any other designed ankyrin repeat domain, comprisedin said recombinant binding protein, or, more preferably, wherein saidtwo designed ankyrin repeat domains with binding specificity for serumalbumin are one N-terminal and one C-terminal of any other proteindomain, preferably any other designed ankyrin repeat domain, comprisedin said recombinant binding protein. Preferably, said two designedankyrin repeat domains with binding specificity for serum albumin arenot both C-terminal of any other protein domain, preferably any otherdesigned ankyrin repeat domain, comprised in said recombinant bindingprotein. Examples of different arrangements of designed ankyrin repeatdomains within a recombinant binding protein are given in SEQ ID NOs: 95to 179, and are described in the Examples. SEQ ID NOs: 134 illustratesthe preferred arrangement of the two designed ankyrin repeat domainswith binding specificity for serum albumin in a recombinant bindingprotein of the present invention.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least three, preferably comprising at least four,more preferably comprising four designed ankyrin repeat domains, whereintwo of said at least three, preferably at least four, more preferablyfour designed ankyrin repeat domains each have binding specificity forserum albumin, and/or, preferably and wherein said at least three,preferably at least four, more preferably four designed ankyrin repeatdomains are linked by polypeptide linkers.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said designed ankyrinrepeat domains with binding specificity for serum albumin each have atleast 90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99% or 100% identitywith any amino acid sequence selected from the group of SEQ ID NOs: 44to 52, preferably SEQ ID NOs: 48 to 50, more preferably SEQ ID NOs: 49and 50, more preferably SEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said designed ankyrinrepeat domains with binding specificity for serum albumin are selectedfrom any amino acid sequence selected from the group of SEQ ID NOs: 44to 52, preferably SEQ ID NOs: 48 to 50, more preferably SEQ ID NOs: 49and 50, more preferably SEQ ID NO: 50, and wherein in each of said twodesigned ankyrin repeat domains with binding specificity for serumalbumin up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 amino acids areexchanged by any amino acid.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said designed ankyrinrepeat domains with binding specificity for serum albumin are selectedfrom any amino acid sequence selected from the group of SEQ ID NOs: 44to 52, preferably SEQ ID NOs: 48 to 50, more preferably SEQ ID NOs: 49and 50, more preferably SEQ ID NO: 50. In one embodiment, the inventionrelates to a recombinant binding protein comprising at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin, wherein said designed ankyrin repeat domains with bindingspecificity for serum albumin each comprise the amino acid sequence ofSEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said designed ankyrinrepeat domains with binding specificity for serum albumin are at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical in amino acid sequence. In one embodiment,said designed ankyrin repeat domains with binding specificity for serumalbumin of said recombinant binding protein are identical in amino acidsequence. For example, the two designed ankyrin repeat domains withbinding specificity for serum albumin comprised in SEQ ID NO: 130 are atleast 95% identical (6 residues difference on 124 amino acids). Inanother example, the two designed ankyrin repeat domains with bindingspecificity for serum albumin comprised in SEQ ID NO: 129 are at least80% identical (24 residues difference on 124 amino acids).

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein the designed ankyrinrepeat domains with binding specificity for serum albumin are able tosimultaneously bind one serum albumin molecule each. Preferably, saidserum albumin is of human origin. Examples for simultaneous binding oftwo human serum albumin molecules by recombinant binding proteins of thepresent invention, comprising two designed ankyrin repeat domains withbinding specificity for serum albumin, are shown in Example 7.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least 3, 4, 5, 6, 7, 8, 9, 10 designed ankyrinrepeat domains with binding specificity for serum albumin.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least three, preferably at least four, morepreferably comprising four, designed ankyrin repeat domains, wherein atleast two, more preferably two, of said designed ankyrin repeat domainsare designed ankyrin repeat domains with binding specificity for serumalbumin, more preferably human serum albumin, and wherein said at leasttwo, more preferably two, designed ankyrin repeat domains with bindingspecificity for serum albumin are at least, preferably are, oneN-terminal and one C-terminal of any other designed ankyrin repeatdomain, preferably the other two designed ankyrin repeat domains, andwherein said at least two, more preferably two, designed ankyrin repeatdomains with binding specificity for serum albumin are each bindingserum albumin, preferably serum albumin of human origin, in PBS with adissociation constant (Kd) of at least 10⁻⁵M, preferably below 10⁻⁶M, ormore preferably below 10⁻⁷M, and wherein said recombinant bindingprotein exhibits an increased terminal half-life, preferably anincreased terminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, or 50% compared to the recombinant binding proteincomprising only one designed ankyrin repeat domain with bindingspecificity for serum albumin, and wherein said at least three,preferably at least four, more preferably four, designed ankyrin repeatdomains are linked by polypeptide linkers. In one embodiment, said atleast two, more preferably two, designed ankyrin repeat domains withbinding specificity for serum albumin of said recombinant bindingprotein are at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, more preferablyidentical.

In one embodiment, protein domains or the designed ankyrin repeatdomains present in a recombinant binding protein of the presentinvention are linked by polypeptide linkers of any amino acid sequencecomposition. In one embodiment, the polypeptide linkers linking proteindomains or the designed ankyrin repeat domains present in a recombinantbinding protein of the present invention comprise amino acid sequencesselected from the group consisting of amino acid sequences SEQ ID NOs: 2to 9, more preferably SEQ ID NOs: 3 to 9, more preferably SEQ ID NOs: 4to 9, more preferably SEQ ID NOs: 6 or 9, more preferably SEQ ID NO: 9,in which up to 4, 3, 2, 1, 0 amino acids are exchanged by any aminoacid. In one embodiment, said polypeptide linkers comprise an amino acidsequence chosen from any of amino acid sequences SEQ ID NOs: 2 to 9,more preferably SEQ ID NOs: 3 to 9, more preferably SEQ ID NOs: 4 to 9,more preferably SEQ ID NOs: 6 or 9, more preferably SEQ ID NO: 9. In oneembodiment, the flanking N-terminal Gly Ser of SEQ ID NOs: 7 to 9 and/orthe flanking C-terminal Gly Ser of SEQ ID NOs: 2 to 9 are optionallymissing. In one embodiment, SEQ ID NOs: 7 to 9 additionally comprisesArg Ser C-terminally (as e.g. present in SEQ ID NOs: 68 and 109). In oneembodiment, the second-to-C-terminal amino acid glycine of saidpolypeptide linkers of SEQ ID NOs: 2 to 6 may be exchanged by arginine(as e.g. present in SEQ ID NOs: 70 and 88). In one embodiment, thepolypeptide linkers linking the designed ankyrin repeat domains presentin a recombinant binding protein of the present invention consist of anamino acid sequence selected from of any of amino acid sequences SEQ IDNOs: 2 to 9, more preferably SEQ ID NOs: 3 to 9, more preferably SEQ IDNOs: 4 to 9, more preferably SEQ ID NOs: 6 or 9, more preferably SEQ IDNO: 9. In one embodiment, said polypeptide linkers present in arecombinant binding protein of the present invention are 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical, preferably identical.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said polypeptide linkerscomprise amino acid sequences selected from the group consisting ofamino acid sequences SEQ ID NOs: 2 to 9, more preferably SEQ ID NOs: 3to 9, more preferably SEQ ID NOs: 4 to 9, more preferably SEQ ID NOs: 6or 9, more preferably SEQ ID NO: 9, in which up to 4, 3, 2, 1, 0 aminoacids are exchanged by any amino acid, and wherein said designed ankyrinrepeat domains with binding specificity for serum albumin each have atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identitywith any amino acid sequence selected from the group of SEQ ID NOs: 44to 52, preferably SEQ ID NOs: 48 to 50, more preferably SEQ ID NOs: 49and 50, more preferably SEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said polypeptide linkersconsist of an amino acid sequences selected from the amino acidsequences SEQ ID NOs: 6 or 9, in which up to 4, 3, 2, 1, 0 amino acidsare exchanged by any amino acid, and wherein said designed ankyrinrepeat domains with binding specificity for serum albumin each consistof an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100% identity with any amino acid sequenceselected from the group of SEQ ID NOs: 48 to 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin each comprise the amino acid sequence of SEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin each comprise the amino acid sequence of SEQ ID NO: 50, andwherein said recombinant binding protein exhibits improved storagestability, preferably reduced amounts of degradation products afterstorage at 40° C. for 1 month at 10 mg/ml in PBS, compared to therecombinant binding protein, wherein said at least two designed ankyrinrepeat domains with binding specificity for serum albumin each comprisethe amino acid sequence of SEQ ID NO: 49, and/or compared to therecombinant binding protein, wherein said at least two designed ankyrinrepeat domains with binding specificity for serum albumin each comprisethe amino acid sequence of SEQ ID NO: 51, preferably compared to therecombinant binding protein, wherein said at least two designed ankyrinrepeat domains with binding specificity for serum albumin each comprisethe amino acid sequence of SEQ ID NO: 49.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin each comprise the amino acid sequence of SEQ ID NO: 50, andwherein said designed ankyrin repeat domains are linked by polypeptidelinkers each comprising the amino acid sequence of SEQ ID NO: 9.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin each consist of the amino acid sequence of SEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least two designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said at least twodesigned ankyrin repeat domains with binding specificity for serumalbumin each consist of the amino acid sequence of SEQ ID NO: 50, andwherein said designed ankyrin repeat domains are linked by polypeptidelinkers each consisting of the amino acid sequence of SEQ ID NO: 9.

In one embodiment the invention relates to a recombinant binding proteincomprising four designed ankyrin repeat domains, wherein two of saiddesigned ankyrin repeat domains are designed ankyrin repeat domains withbinding specificity for serum albumin, wherein said two designed ankyrinrepeat domains with binding specificity for serum albumin each comprisethe amino acid sequence of SEQ ID NO: 50, and wherein said designedankyrin repeat domains are linked by polypeptide linkers each comprisingthe amino acid sequence of SEQ ID NO: 9, and wherein said designedankyrin repeat domains are arranged (from N-terminal side to C-terminalside): SEQ ID NO: 50-SEQ ID NO: 9-XXX-SEQ ID NO: 9-YYY-SEQ ID NO: 9-SEQID NO: 50, wherein XXX and YYY each represent a designed ankyrin repeatdomain with binding specificity for another target than serum albumin.

In one embodiment, the present invention relates to a recombinantbinding protein comprising at least a first, a second, a third, and afourth designed ankyrin repeat domain, wherein said first designedankyrin repeat domain has binding specificity for VEGF-A, and whereinsaid second designed ankyrin repeat domain has binding specificity forHGF, and wherein said third and fourth designed ankyrin repeat domainseach have binding specificity for serum albumin. Preferably, saidrecombinant binding protein consists of a single polypeptide chain. Morepreferably, said first, second, third and fourth designed ankyrin repeatdomain are linked by polypeptide linkers. In one embodiment, the presentinvention relates to a recombinant binding protein comprising a first, asecond, a third, and a fourth designed ankyrin repeat domain, whereinsaid first designed ankyrin repeat domain has binding specificity forVEGF-A, and wherein said second designed ankyrin repeat domain hasbinding specificity for HGF, and wherein said third and fourth designedankyrin repeat domains each have binding specificity for serum albumin.Examples of such recombinant binding proteins are given in amino acidsequences SEQ ID NOs: 95 to 108 and 116 to 179.

Preferably, the designed ankyrin repeat domain with binding specificityfor VEGF-A binds VEGF-A of mouse, rat, dog, rabbit, cynomolgus monkey,or human origin, more preferably VEGF-A of mouse, cynomolgus monkey orhuman origin, more preferably VEGF-A of cynomolgus monkey or humanorigin, more preferably VEGF-A of human origin. Preferably, VEGF-A ishuman VEGF-A165. Examples of designed ankyrin repeat domains withbinding specificity to VEGF-A are given herein (SEQ ID NOs: 12 to 21;see examples) and further examples are described in WO 2010/060748 andWO 2011/135067.

Preferably, the designed ankyrin repeat domain with binding specificityfor HGF binds HGF of mouse, rat, dog, rabbit, cynomolgus monkey, orhuman origin, more preferably HGF of mouse, cynomolgus monkey or humanorigin, more preferably HGF of cynomolgus monkey or human origin, morepreferably HGF of human origin. Examples of designed ankyrin repeatdomains with binding specificity to HGF are given herein (SEQ ID NOs: 23to 37; see examples) and further examples are described in WO2014/191574.

In one embodiment the recombinant binding protein or designed ankyrinrepeat domain is devoid of a free Cys residue. A “free Cys residue” isnot involved in the formation of a disulfide bond. In one embodiment,the invention relates to a binding protein or binding domain free of anyCys residue. In one embodiment, the designed ankyrin repeat domainand/or recombinant binding protein devoid of any disulfide bond. Thedisulfide bonds of antibody fragments for example are known to theperson skilled in the art to hamper the simple production of theantibody fragments in bacteria.

The techniques to modify a recombinant binding protein of the presentinvention are well known to the person skilled in the art.

In particular, the invention relates to a recombinant binding proteincomprising at least a first, a second, a third, and a fourth designedankyrin repeat domain, wherein said first designed ankyrin repeat domainbinds VEGF-A in PBS with a dissociation constant (Kd) below 10⁻⁷M;preferably below 10⁻⁸M; more preferably below 10⁻⁹M; or more preferablybelow 10⁻¹⁰M; and wherein said second designed ankyrin repeat domainbinds HGF in PBS with a Kd below 10⁻⁷M; preferably below 10⁻⁸M; morepreferably below 10⁻⁹M; or more preferably below 10⁻¹⁰M; and whereinsaid third and fourth designed ankyrin repeat domains each bind serumalbumin in PBS with a Kd below 10⁻⁵M; preferably below 10⁻⁶M; or morepreferably below 10⁻⁷M. Examples of designed ankyrin repeat domains withbinding specificity to VEGF-A, designed ankyrin repeat domains withbinding specificity to HGF, and designed ankyrin repeat domains withbinding specificity to serum albumin are given herein (SEQ ID NOs: 12 to56; see examples).

Furthermore, the invention relates to a recombinant binding proteincomprising at least a first, a second, a third, and a fourth designedankyrin repeat domain, wherein said first designed ankyrin repeat domaininhibits the binding of human VEGF-A to human VEGFR-2 in PBS with anIC₅₀ value below 10⁻⁷M, preferably 10⁻⁸M, more preferably 10⁻⁹M, andwherein said second designed ankyrin repeat domain inhibits the bindingof human HGF to human cMet in PBS with an IC₅₀ value below 10⁻⁷M,preferably 10⁻⁸M, more preferably 10⁻⁹M. Different examples of designedankyrin repeat domains selected from SEQ ID NOs: 12 to 37 are given inthe examples.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain comprises an amino acid sequence that has at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequenceidentity with one designed ankyrin repeat domain selected from the groupconsisting of amino acid sequences SEQ ID NOs: 12 to 21, more preferablySEQ ID NOs: 17 to 21, more preferably SEQ ID NOs: 18 to 20, morepreferably SEQ ID NO: 18. In one embodiment, said first designed ankyrinrepeat domain comprises an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 12 to 21, more preferablySEQ ID NOs: 17 to 21, more preferably SEQ ID NOs: 18 to 20, morepreferably SEQ ID NO: 18, and amino acid sequences in which up to 10, 9,8, 7, 6, 5, 4, 3, 2, 1, 0 amino acids of SEQ ID NOs: 12 to 21, morepreferably SEQ ID NOs: 17 to 21, more preferably SEQ ID NOs: 18 to 20,more preferably SEQ ID NO: 18, are exchanged by any amino acid. In oneembodiment, said first designed ankyrin repeat domain comprises an aminoacid sequence selected from the group consisting of amino acid sequencesSEQ ID NOs: 14 to 21, in which individual amino acids are replaced byany amino acid occurring at the same position of an alignment of theamino acid sequences of SEQ ID NOs: 14 to 21. In one embodiment, saidfirst designed ankyrin repeat domain comprises an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NOs:12 to 21, more preferably SEQ ID NOs: 17 to 21, more preferably SEQ IDNOs: 18 to 20, more preferably SEQ ID NO: 18. In one embodiment, saidfirst designed ankyrin repeat domain consists of an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NOs:12 to 21, more preferably SEQ ID NOs: 17 to 21, more preferably SEQ IDNOs: 18 to 20, more preferably SEQ ID NO: 18. Furthermore, said seconddesigned ankyrin repeat domain of said recombinant binding proteinpreferably comprises an amino acid sequence that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequenceidentity with one designed ankyrin repeat domain selected from the groupconsisting of amino acid sequences SEQ ID NOs: 23 to 37, more preferablySEQ ID NOs: 23 to 27, more preferably SEQ ID NOs: 25 to 27, morepreferably SEQ ID NO: 26. In one embodiment, said second designedankyrin repeat domain comprises an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NOs: 23 to 37, morepreferably SEQ ID NOs: 23 to 27, more preferably SEQ ID NOs: 25 to 27,more preferably SEQ ID NO: 26, and amino acid sequences in which up to10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0 amino acids of SEQ ID NOs: 23 to 37,more preferably SEQ ID NOs: 23 to 27, more preferably SEQ ID NOs: 25 to27, more preferably SEQ ID NO: 26, are exchanged by any amino acid. Inone embodiment, said second designed ankyrin repeat domain comprises anamino acid sequence selected from the group consisting of amino acidsequences SEQ ID NOs: 23 to 27, in which individual amino acids arereplaced by any amino acid occurring at the same position of analignment of the amino acid sequences of SEQ ID NOs: 23 to 27. In oneembodiment, said second designed ankyrin repeat domain comprises anamino acid sequence selected from the group consisting of amino acidsequences SEQ ID NOs: 23 to 37, more preferably SEQ ID NOs: 23 to 27,more preferably SEQ ID NOs: 25 to 27, more preferably SEQ ID NO: 26. Inone embodiment, said second designed ankyrin repeat domain consists ofan amino acid sequence selected from the group consisting of amino acidsequences SEQ ID NOs: 23 to 37, more preferably SEQ ID NOs: 23 to 27,more preferably SEQ ID NOs: 25 to 27, more preferably SEQ ID NO: 26. Inone embodiment, said third and fourth designed ankyrin repeat domains ofsaid recombinant binding protein each comprise an amino acid sequencethat has at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99%, 100%amino acid sequence identity with one ankyrin repeat domain selectedfrom the group consisting of amino acid sequences SEQ ID NOs: 40 to 56,preferably SEQ ID NOs: 48 to 52, more preferably SEQ ID NOs: 48 to 50,more preferably SEQ ID NO: 50. In one embodiment, said third and fourthdesigned ankyrin repeat domains each comprise an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NOs:40 to 56, preferably SEQ ID NOs: 48 to 52, more preferably SEQ ID NOs:48 to 50, more preferably SEQ ID NO: 50, and amino acid sequences inwhich up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0 amino acids of SEQ ID NOs:40 to 56, preferably SEQ ID NOs: 48 to 52, more preferably SEQ ID NOs:48 to 50, more preferably SEQ ID NO: 50, are exchanged by any otheramino acid. In one embodiment, said third and fourth designed ankyrinrepeat domains each comprise an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NOs: 42 to 51, in whichindividual amino acids are replaced by any amino acid occurring at thesame position of an alignment of the amino acid sequences of SEQ ID NOs:42 to 51. In one embodiment, said third and fourth designed ankyrinrepeat domains each comprise an amino acid sequence selected from thegroup consisting of amino acid sequences SEQ ID NOs: 40 to 56,preferably SEQ ID NOs: 48 to 52, more preferably SEQ ID NOs: 48 to 50,more preferably SEQ ID NO: 50. In one embodiment, said third and fourthdesigned ankyrin repeat domains each consist of an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NOs:40 to 56, preferably SEQ ID NOs: 48 to 52, more preferably SEQ ID NOs:48 to 50, more preferably SEQ ID NO: 50. In one embodiment, said thirdand fourth designed ankyrin repeat domains are identical. Further inthis embodiment, said designed ankyrin repeat domains are linked bypolypeptide linkers selected from the group consisting of amino acidsequences SEQ ID NOs: 2 to 9, more preferably SEQ ID NOs: 3 to 9, morepreferably SEQ ID NOs: 4 to 9, more preferably SEQ ID NOs: 6 or 9, morepreferably SEQ ID NO: 9, and amino acid sequences in which up to 4, 3,2, 1, 0 amino acids of SEQ ID NOs: 2 to 9, more preferably SEQ ID NOs: 3to 9, more preferably SEQ ID NOs: 4 to 9, more preferably SEQ ID NOs: 6or 9, more preferably SEQ ID NO: 9, are exchanged by any amino acid. Inone embodiment, said polypeptide linkers comprise an amino acid sequenceselected from the group consisting of amino acid sequences SEQ ID NOs: 2to 9, more preferably SEQ ID NOs: 3 to 9, more preferably SEQ ID NOs: 4to 9, more preferably SEQ ID NOs: 6 or 9, more preferably SEQ ID NO: 9.In one embodiment, the flanking N-terminal Gly Ser of SEQ ID NOs: 7 to 9and/or the flanking C-terminal Gly Ser of SEQ ID NOs: 2 to 9 areoptionally missing. In one embodiment, SEQ ID NOs: 7 to 9 additionallycomprises Arg Ser C-terminally (as e.g. present in SEQ ID NOs: 97 and98). In one embodiment, the second-to-C-terminal amino acid glycine ofsaid polypeptide linkers of SEQ ID NOs: 2 to 6 may be exchanged byarginine (as e.g. present in SEQ ID NOs: 99 and 100). In one embodiment,the polypeptide linkers linking the designed ankyrin repeat domainspresent in a recombinant binding protein of the present inventionconsist of an amino acid sequence selected from of any of amino acidsequences SEQ ID NOs: 2 to 9, more preferably SEQ ID NOs: 3 to 9, morepreferably SEQ ID NOs: 4 to 9, more preferably SEQ ID NOs: 6 or 9, morepreferably SEQ ID NO: 9. In one embodiment, said polypeptide linkerspresent in a recombinant binding protein of the present invention areidentical. Examples of such polypeptide linkers, variations thereof, andthe use of such polypeptide linkers in recombinant binding proteins aregiven in the examples.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain comprises an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 12 to 21, preferably SEQID NOs: 17 to 21, more preferably SEQ ID NOs: 18 to 20, more preferablySEQ ID NO: 18, and wherein said second designed ankyrin repeat domaincomprises an amino acid sequence selected from the group consisting ofamino acid sequences SEQ ID NOs: 23 to 37, more preferably SEQ ID NOs:23 to 27, more preferably SEQ ID NOs: 25 to 27, more preferably SEQ IDNO: 26, and wherein said third and fourth designed ankyrin repeatdomains each comprise an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 40 to 56, preferably SEQID NOs: 48 to 52, more preferably SEQ ID NOs: 48 to 50, more preferablySEQ ID NO: 50, and wherein said designed ankyrin repeat domains arelinked by polypeptide linkers selected from the group consisting ofamino acid sequences SEQ ID NOs: 2 to 9, more preferably SEQ ID NOs: 3to 9, more preferably SEQ ID NOs: 4 to 9, more preferably SEQ ID NOs: 6or 9, more preferably SEQ ID NO: 9.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain has binding specificity for VEGF-A, and wherein saidsecond designed ankyrin repeat domain has binding specificity for HGF,and wherein said third and fourth designed ankyrin repeat domains eachhave binding specificity for serum albumin, and wherein said designedankyrin repeat domains are linked by polypeptide linkers, and whereinsaid recombinant binding protein can bind VEGF-A and HGF, morepreferably VEGF-A, HGF, and serum albumin, more preferably human VEGF-A,human HGF and human serum albumin, simultaneously. In one embodiment,said recombinant binding protein can bind two serum albumin molecules,more preferably two human serum albumin molecules, simultaneously.

The terms “first, “second”, “third”, and optionally “fourth”, used in“first designed ankyrin repeat domain”, “second designed ankyrin repeatdomain”, “third designed ankyrin repeat domain”, and “fourth designedankyrin repeat domain”, do not indicate or imply any positionalarrangement of said designed ankyrin repeat domains within therecombinant binding protein.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain which are linked by polypeptide linkers.In one embodiment, said first designed ankyrin repeat domain isN-terminal of the C-terminal designed ankyrin repeat domain andC-terminal of the other two designed ankyrin repeat domains. In oneembodiment, said second designed ankyrin repeat domain, having a bindingspecificity for HGF, is C-terminal of the N-terminal designed ankyrinrepeat domain and N-terminal of the other two designed ankyrin repeatdomains. In one embodiment, said third and fourth designed ankyrinrepeat domains, each having a binding specificity for serum albumin, areone N-terminal and one C-terminal of the other two designed ankyrinrepeat domains, or they are N-terminal of the other two designed ankyrinrepeat domains, more preferably said third and fourth designed ankyrinrepeat domains are one N-terminal and one C-terminal of the other twodesigned ankyrin repeat domains. In one embodiment, said third designedankyrin repeat domain is N-terminal of the other three designed ankyrinrepeat domains, said fourth designed ankyrin repeat domain is C-terminalof the other three designed ankyrin repeat domains, said second designedankyrin repeat domain is C-terminal of said third designed ankyrinrepeat domain and N-terminal of said first designed ankyrin repeatdomain, and said first designed ankyrin repeat domain is C-terminal ofsaid second designed ankyrin repeat domain and N-terminal of said fourthdesigned ankyrin repeat domain.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain has binding specificity for VEGF-A, and wherein saidsecond designed ankyrin repeat domain has binding specificity for HGF,and wherein said third and fourth designed ankyrin repeat domains eachhave binding specificity for serum albumin, and wherein said designedankyrin repeat domains are linked by polypeptide linkers. In oneembodiment, said first, second, third and fourth designed ankyrin repeatdomains are in the order (from N terminus to C terminus)third-second-first-fourth, third-fourth-second-first,fourth-second-first-third, or fourth-third-second-first, even morepreferably third-second-first-fourth, or fourth-second-first-third, morepreferably third-second-first-fourth.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain has binding specificity for VEGF-A, and wherein saidsecond designed ankyrin repeat domain has binding specificity for HGF,and wherein said third and fourth designed ankyrin repeat domains eachhave binding specificity for serum albumin, wherein said recombinantbinding protein binds VEGF-A, preferably human VEGF-A, with an EC₅₀ ofless than 10⁻⁷M, preferably less than 10⁻⁸ M, more preferably less than10⁻⁹M, more preferably less than 10⁻¹⁰M.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said third and fourth designedankyrin repeat domains each comprise the amino acid sequence of SEQ IDNO: 50. In one embodiment, the invention relates to a recombinantbinding protein comprising at least a first, a second, a third, and afourth designed ankyrin repeat domain, wherein said first designedankyrin repeat domain comprises the amino acid sequence of SEQ ID NO:18, and wherein said second designed ankyrin repeat domain comprises theamino acid sequence of SEQ ID NO: 26, and wherein said third and fourthdesigned ankyrin repeat domains each comprise the amino acid sequence ofSEQ ID NO: 50, and wherein said designed ankyrin repeat domains arelinked by polypeptide linkers each comprising the amino acid sequence ofSEQ ID NO: 9.

In one embodiment the invention relates to a recombinant binding proteincomprising at least a first, a second, a third, and a fourth designedankyrin repeat domain, wherein said first designed ankyrin repeat domaincomprises the amino acid sequence of SEQ ID NO: 18, and wherein saidsecond designed ankyrin repeat domain comprises the amino acid sequenceof SEQ ID NO: 26, and wherein said third and fourth designed ankyrinrepeat domains each comprise the amino acid sequence of SEQ ID NO: 50,and wherein said designed ankyrin repeat domains are linked bypolypeptide linkers each comprising the amino acid sequence of SEQ IDNO: 9, and wherein said designed ankyrin repeat domains are arranged(from N-terminal side to C-terminal side): SEQ ID NO: 50-SEQ ID NO:9-SEQ ID NO: 26-SEQ ID NO: 9-SEQ ID NO: 18-SEQ ID NO: 9-SEQ ID NO: 50.

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain has binding specificity for VEGF-A, and wherein saidsecond designed ankyrin repeat domain has binding specificity for HGF,and wherein said third and fourth designed ankyrin repeat domains eachhave binding specificity for serum albumin, and wherein said designedankyrin repeat domains are linked by polypeptide linkers each comprisingthe amino acid sequence of SEQ ID NO: 9, and wherein said recombinantbinding protein binds VEGF-A, and/or HGF, preferably VEGF-A with alower, i.e. better, EC₅₀ compared to the recombinant binding proteinwherein said designed ankyrin repeat domains are linked by polypeptidelinkers each comprising the amino acid sequence SEQ ID NO: 6. Examplesof the influence of the linker on EC₅₀ are given in Example 8 and theterm “lower EC₅₀” is well known to the person skilled in the art.Preferably, the term “lower EC₅₀” means an EC₅₀ value which is improvedby a factor of 1.1, more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0.

In one embodiment the invention relates to a recombinant binding proteincomprising at least a first, a second, a third, and a fourth designedankyrin repeat domain, wherein said first designed ankyrin repeat domainhas binding specificity for VEGF-A, and wherein said second designedankyrin repeat domain has binding specificity for HGF, and wherein saidthird and fourth designed ankyrin repeat domains each have bindingspecificity for serum albumin, wherein said recombinant binding proteinbinds VEGF-A, and/or HGF, preferably VEGF-A with a lower, i.e. better,EC₅₀ compared to the recombinant binding protein comprising only onedesigned ankyrin repeat domain with binding specificity for serumalbumin. Examples are given in Example 8.

In one embodiment, the invention relates to a recombinant bindingprotein comprising an amino acid sequence that has at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequenceidentity with an amino acid sequence selected from the group consistingof amino acid sequences SEQ ID NOs: 134 to 179, preferably SEQ ID NOs:134 to 158, more preferably SEQ ID NOs: 134 to 149, more preferably SEQID NOs: 134 to 140, more preferably SEQ ID NO: 134.

In one embodiment, the invention relates to a recombinant bindingprotein comprising an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 134 to 179, preferablySEQ ID NOs: 134 to 158, more preferably SEQ ID NOs: 134 to 149, morepreferably SEQ ID NOs: 134 to 140, more preferably SEQ ID NO: 134, inwhich up to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 aminoacids are exchanged by any amino acid.

In any embodiment of the present invention relating to a designedankyrin repeat domain or a recombinant binding protein comprising anamino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to a given amino acid sequence, thenon-identical amino acids may be located at any position of the designedankyrin repeat domain or the recombinant binding protein.

Likewise, in any embodiment of the present invention relating to adesigned ankyrin repeat domain or a recombinant binding protein in whichup to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acidsare exchanged by any amino acid, the exchanged amino acids amino acidsmay be located at any position of the designed ankyrin repeat domain.

In one embodiment, the invention relates to a recombinant bindingprotein comprising an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 134 to 179, preferablySEQ ID NOs: 134 to 158, more preferably SEQ ID NOs: 134 to 149, morepreferably SEQ ID NOs: 134 to 140, more preferably SEQ ID NO: 134.

The invention particularly relates to a recombinant binding proteincomprising an amino acid sequence that has at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity withthe amino acid sequence of SEQ ID NO: 134.

In one embodiment, the invention relates to a recombinant bindingprotein comprising the amino acid sequence of SEQ ID NO: 134, in whichup to 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acidsare exchanged by any amino acid.

The invention further particularly relates to a recombinant bindingprotein comprising the amino acid sequence consisting of the amino acidsequence of SEQ ID NO: 134. In one embodiment, the invention relates toa recombinant binding protein comprising the amino acid sequence of SEQID NO: 134.

In one embodiment, the invention relates to a recombinant bindingprotein consisting of an amino acid sequence selected from the groupconsisting of amino acid sequences SEQ ID NOs: 134 to 179, preferablySEQ ID NOs: 134 to 158, more preferably SEQ ID NOs: 134 to 149, morepreferably SEQ ID NOs: 134 to 140, more preferably SEQ ID NO: 134.

Preferred is SEQ ID NO: 134. Preferred is a recombinant binding protein,wherein the amino acid sequence is SEQ ID NO: 134. Preferred is aprotein, wherein the amino acid sequence is SEQ ID NO: 134. Preferred isa recombinant binding protein consisting of the amino acid sequence ofSEQ ID NO: 134.

Multiple features make SEQ ID NO: 134 the preferred recombinant bindingprotein of the invention. It comprises two designed ankyrin repeatdomains with binding specificity for serum albumin each consisting ofSEQ ID NO: 50, which shows improved storage stability properties (seeExample 9; FIG. 2) compared to known designed ankyrin repeat domain withbinding specificity for serum albumin. It comprises two designed ankyrinrepeat domains with binding specificity for serum albumin, whichsurprisingly leads to improved pharmacokinetic properties (Examples 5and 6, FIGS. 3A-3D and FIGS. 4A-4B). The two designed ankyrin repeatdomains with binding specificity for serum albumin are flanking theother designed ankyrin repeat domains leading to the bestpharmacokinetic properties observed (Example 6). The designed ankyrinrepeat domains and with binding specificity for VEGF-A and HGF as wellas their structural arrangement were chosen to maximize activity of thecompound (Example 8). The designed ankyrin repeat domains are connectedusing a PT-rich linker, surprisingly leading to improved activity of theindividual designed ankyrin repeat domains (Example 8) and surprisinglyleading to improved pharmacokinetic properties (Example 5).

In one embodiment, the invention relates to a recombinant bindingprotein comprising at least a first, a second, a third, and a fourthdesigned ankyrin repeat domain, wherein said first designed ankyrinrepeat domain has binding specificity for VEGF-A, and wherein saidsecond designed ankyrin repeat domain has binding specificity for HGF,and wherein said third and fourth designed ankyrin repeat domains eachhave binding specificity for serum albumin, and wherein said first,second, third, and fourth designed ankyrin repeat domain are linked bypolypeptide linkers, and wherein said recombinant binding proteinexhibits an increase in terminal half-life, preferably an increase interminal half-life of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or45%, compared to the recombinant binding protein lacking said fourthdesigned ankyrin repeat domain with binding specificity for serumalbumin. Examples of such an increase in terminal half-life are given inExamples 5 and 6 and FIGS. 3A-3D and FIGS. 4A-4B.

In one embodiment, the invention relates to a nucleic acid encoding theamino acid sequence of a designed ankyrin repeat domain or a recombinantbinding protein of the present invention, more preferably a recombinantbinding protein of the present invention. In one embodiment, theinvention relates to a nucleic acid encoding the amino acid sequence ofany recombinant binding protein of the present invention comprising atleast two, more preferably comprising two, designed ankyrin repeatdomains with binding specificity for serum albumin. In one embodiment,the invention relates to a nucleic acid encoding the amino acid sequenceof a recombinant binding protein of the present invention. Furthermore,the invention relates to vectors comprising any nucleic acid of theinvention. Nucleic acids are well known to the skilled person. In theexamples, nucleic acids were used to produce designed ankyrin repeatdomains or recombinant binding proteins of the invention in E. coli.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a recombinant binding protein and/or a designed ankyrinrepeat domain of the present invention, or a nucleic acid encoding arecombinant binding protein and/or a designed ankyrin repeat domain ofthe present invention, and optionally a pharmaceutically acceptablecarrier and/or diluent.

In one embodiment, the invention relates to a pharmaceutical compositioncomprising a recombinant binding protein or a nucleic acid encoding arecombinant binding protein, and optionally a pharmaceuticallyacceptable carrier and/or diluent.

Pharmaceutical acceptable carriers and/or diluents are known to theperson skilled in the art and are explained in more detail below. Evenfurther, a diagnostic composition comprising one or more of the abovementioned recombinant binding proteins and/or designed ankyrin repeatdomains, and/or nucleic acids, in particular recombinant bindingproteins, is considered.

A pharmaceutical composition comprises a recombinant binding protein,and/or a designed ankyrin repeat domain, and/or a nucleic acid asdescribed herein and a pharmaceutically acceptable carrier, excipient orstabilizer, for example as described in Remington's PharmaceuticalSciences 16^(th) edition, Osol, A. Ed., 1980. Suitable carriers,excipients or stabilizers known to the skilled man are saline, Ringer'ssolution, dextrose solution, Hank's solution, fixed oils, ethyl oleate,5% dextrose in saline, substances that enhance isotonicity and chemicalstability, buffers and preservatives. Other suitable carriers includeany carrier that does not itself induce the production of antibodiesharmful to the individual receiving the composition such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids and amino acid copolymers. A pharmaceutical composition may alsobe a combination formulation, comprising an additional active agent,such as an anti-cancer agent or an anti-angiogenic agent, or anadditional bioactive compound.

One embodiment of the present invention relates to the use of arecombinant binding protein of the present invention comprising at leasttwo, preferably comprising two, designed ankyrin repeat domains withbinding specificity for serum albumin for manufacturing a pharmaceuticalcomposition, wherein said recombinant binding protein exhibits anincreased terminal half-life, preferably an increased terminal half-lifeof at least 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250%, compared to therecombinant binding protein comprising only one designed ankyrin repeatdomain with binding specificity for serum albumin.

In one embodiment, a pharmaceutical composition comprises at least onerecombinant binding protein as described herein and a detergent such asnonionic detergent, a buffer such as phosphate, and a sugar such assucrose. In one embodiment, such a composition comprises recombinantbinding proteins as described above and PBS.

In one embodiment, the invention relates to the use of a pharmaceuticalcomposition, or a recombinant binding protein according to the presentinvention for the treatment of a disease. For that purpose, thepharmaceutical composition, or the recombinant binding protein accordingto the present invention is administered, to a patient in need thereof,in a therapeutically effective amount. Administration may includetopical administration, oral administration, and parenteraladministration. The typical route of administration is parenteraladministration. In parental administration, the medicament of thisinvention will be formulated in a unit dosage injectable form such as asolution, suspension or emulsion, in association with thepharmaceutically acceptable excipients as defined above. The dosage andmode of administration will depend on the individual to be treated andthe particular disease.

Further, any of the above mentioned pharmaceutical composition orrecombinant binding protein is considered for the treatment of adisorder.

A pharmaceutical composition of the present invention may beadministered by e.g. parenteral administration. In parentaladministration, the medicament of this invention will be formulated in aunit dosage injectable form such as a solution, suspension or emulsion,in association with the pharmaceutically acceptable excipients asdefined above. The dosage and mode of administration will depend on theindividual to be treated and the particular disease. In one embodiment,said recombinant binding protein or such other pharmaceuticalcomposition described herein is applied intravenously. For parenteralapplication, the recombinant binding protein or said pharmaceuticalcomposition can be injected as bolus injection or by slow infusion at atherapeutically effective amount.

In one embodiment, the invention relates to a method of treatment of amedical condition, the method comprising the step of administering, to apatient in need of such a treatment, a therapeutically effective amountof a recombinant binding protein of the invention. In one embodiment,the invention relates to a method of treatment of a medical condition,the method comprising the step of administering, to a patient in need ofsuch a treatment, a therapeutically effective amount of a pharmaceuticalcomposition of the invention. Example 14 (FIGS. 10A-10C) illustrates theutility of the use of a recombinant binding protein consisting of SEQ IDNO: 134 for the treatment of cancer. In one embodiment, the inventionrelates to the use of a pharmaceutical composition of the presentinvention for the treatment of a disease. In one embodiment, theinvention relates to a pharmaceutical composition for use in thetreatment of a disease.

A “medical condition” (or disorder) may be one that is characterized byinappropriate angiogenesis. A medical condition may be ahyperproliferative condition. Examples of medical conditions suitablefor treatment include autoimmune disorders, inflammatory disorders,retinopathies (particularly proliferative retinopathies),neurodegenerative disorders, infections, and neoplastic diseases. Any ofthe recombinant binding proteins described herein may be used for thepreparation of a medicament for the treatment of such a disorder,particularly a disorder selected from the group consisting of: anautoimmune disorder, an inflammatory disorder, a retinopathy, and aneoplastic disease. The invention particularly relates to a method oftreating a medical condition, the method comprising the step ofadministering, to a patient in need of such treatment, a therapeuticallyeffective amount of a recombinant binding protein or said pharmaceuticalcomposition of the invention. In some embodiments said medical conditionis a neoplastic disease. The term “neoplastic disease”, as used herein,refers to an abnormal state or condition of cells or tissuecharacterized by rapidly proliferating cell growth or neoplasm. In amore specific meaning, the term relates to cancer. In a more specificmeaning, the term may relate to renal cancer and/or gastric cancerand/or multiple myeloma. The term “therapeutically effective amount”means an amount that is sufficient to produce a desired effect on apatient.

In particular, the invention relates to the treatment of a medicalcondition using a pharmaceutical composition of the present invention,wherein said medical condition is cancer.

The use of a recombinant binding protein of the present invention orsaid pharmaceutical compositions for the treatment of cancer diseasescan also be in combination with any other therapy known in the art. Theterm “use in combination with”, as used herein, shall refer to acoadministration, which is carried out under a given regimen. Thisincludes synchronous administration of the different compounds as wellas time-shifted administration of the different compounds (e.g. compoundA is given once and compound B is given several times thereafter, orvice versa, or both compounds are given synchronously and one of the twois also given at later stages).

The use of a recombinant protein for the treatment of a diseaseincluding pathological angiogenesis is further considered. The term“pathological angiogenesis” refers to the formation and growth of bloodvessels during the maintenance and the progression of several diseasestates.

In a further embodiment, the invention relates to the use of arecombinant binding protein of the invention for the manufacture of amedicament that is used for the treatment of a medical condition,preferably a neoplastic disease, more preferably cancer.

In one embodiment, the invention relates to the use of a pharmaceuticalcomposition of the invention for the manufacture of a medicament that isused for the treatment of a medical condition, which may be a neoplasticdisease, in particular cancer.

The formulations to be used for in vivo administration must be asepticor sterile. This is readily accomplished by filtration through sterilefiltration membranes.

The term “selected from the group consisting of” in connection with asingle choice in this invention has the meaning of that particularchoice. For example, in one embodiment, “the invention relates to arecombinant binding protein comprising an amino acid sequence selectedfrom the group consisting of amino acid sequences SEQ ID NO: 134”, whichhas the meaning “the invention relates to a recombinant binding proteincomprising the amino acid sequence of SEQ ID NO: 134”.

In one embodiment the invention relates to a recombinant binding proteincomprising any of the above mentioned repeat domains. In one embodiment,the invention relates to a recombinant binding protein comprising any ofthe above mentioned SEQ ID NO: 134 to 179.

The invention is not restricted to the particular embodiments describedin the Examples. Other sources may be used and processed following thegeneral outline described below.

A number of documents are cited throughout this specification. Thedisclosure content of these documents is herewith incorporated byreference.

This specification refers to a number of amino acid sequences of theamino acid sequence listing of this specification named“P014_Sequence_Protocol.txt” and the amino acid sequences of thesequence protocol are herewith incorporated by reference.

EXAMPLES

All of the standard materials and reagents disclosed here are known tothose skilled in the art, and are available commercially or can beprepared using well-known techniques.

Materials Chemicals were purchased from Sigma-Aldrich (Switzerland).Oligonucleotides were from Microsynth (Switzerland). Unless statedotherwise, DNA polymerases, restriction enzymes and buffers were fromNew England Biolabs (USA) or Thermo Fisher Scientific Fermentas(Lithuania). The cloning and protein production strain was E. coliXL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Recombinant VEGF-A(human, mouse, rat), VEGF-C, PDGF-AB, and HGF (human, cynomolgus monkey,mouse) were from R&D Systems (Biotechne; Minneapolis, USA), Peprotech(Rocky Hill, USA), Sino Biological (Beijing, China), ReliaTech(Wolfenbüttel, Germany) or produced in Chinese Hamster Ovary Cells or inPichia pastoris and purified according to standard protocols. Serumalbumin of different species were from Sigma-Aldrich, InnovativeResearch (Novi, USA), CSL Behring (Switzerland), or collected fromanimals directly using standard methods. Biotinylated VEGF-A or HGF wereobtained chemically via coupling of the biotin moiety to primary aminesof the protein using standard biotinylation reagents and methods (ThermoFisher Scientific Inc., USA). Antibodies were from Thermo FisherScientific or QIAgen (Germany), or were generated using standardimmunization and hybridoma procedures in mice or rabbits, procedureswell known to the person skilled in the relevant art. Cell culturereagents were from Lonza (Switzerland), Roche (Switzerland), ThermoFisher Scientific, and Promocell (Germany).

Molecular Biology

Unless stated otherwise, methods are performed according to describedprotocols (Sambrook J., Fritsch E. F. and Maniatis T., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, NewYork).

Designed Ankyrin Repeat Domains, Libraries and Selections

Methods to generate designed ankyrin repeat protein libraries, examplesof designed ankyrin repeat protein libraries, and methods to selectdesigned ankyrin repeat proteins from libraries of designed ankyrinrepeat proteins are described (WO 2002/020565; WO 2010/060748; WO2012/069654; WO 2012/069655; WO 2014/001442; Binz et al. 2004, loc.cit.).

Example 1 Selection, Expression, Purification, and Analysis of DesignedAnkyrin Repeat Domains with Binding Specificity for VEGF-A, HGF, orSerum Albumin

Using ribosome display (Binz et al., 2004, loc. cit.) designed ankyrinrepeat domains with binding specificity for VEGF-A, HGF, or serumalbumin were selected from combinatorial libraries by methods describedin WO 2010/060748 for the generation of designed ankyrin repeat domainswith binding specificity for VEGF-A, and by methods described in WO2014/191574 for the generation of designed ankyrin repeat domains withbinding specificity for HGF, and by methods described in WO 2012/069654for the generation of designed ankyrin repeat domains with bindingspecificity to serum albumin. The binding of the selected clones towardspecific (VEGF-A, HGF, or serum albumin, respectively) and unspecific(e.g. MBP, E. coli maltose binding protein) targets was assessed bycrude extract ELISA, indicating that hundreds of designed ankyrin repeatdomains with binding specificity to VEGF-A, HGF, or serum albumin,respectively, were successfully selected in each selection for therespective target. For example, the designed ankyrin repeat domains ofSEQ ID NO: 12 to 22 constitute amino acid sequences of ankyrin repeatdomains with binding specificity for VEGF-A, the designed ankyrin repeatdomains of SEQ ID NO: 23 to 37 constitute amino acid sequences ofdesigned ankyrin repeat domains with binding specificity for HGF, andthe designed ankyrin repeat domains of SEQ ID NO: 40 to 56 constituteamino acid sequences of designed ankyrin repeat domains with bindingspecificity for serum albumin.

These designed ankyrin repeat domains with binding specificity forVEGF-A, HGF, or serum albumin, and negative control designed ankyrinrepeat domains with no known binding specificity (i.e. Proteins #10 and#11) were cloned into a pQE (QIAgen, Germany) based expression vectorproviding an N-terminal His-tag to facilitate simple proteinpurification. The proteins were produced and purified with methods knownto the person skilled in the art such as described for example in WO2010/060748.

Example 2 Characterization of Designed Ankyrin Repeat Domains UsingSurface Plasmon Resonance

SPR was measured using a Protein instrument (BioRad) and measurement wasperformed according standard procedures known to the person skilled inthe art. Kd values that were measured for selected proteins are listedin Tables 1 to 3.

TABLE 1 Examples of dissociation constants of designed ankyrin repeatdomains bindind to human VEGF-A Protein #* Kd [pM] 12  94 13  96 16141 * Protein #12, #13, and #16 in this table represent designed ankyrinrepeat domains consisting of the corresponding amino acid sequence ofSEQ ID NO: 12, 13 and 16, and additionally an N-terminal His-tag (SEQ IDNO: 1). Similar VEGF-A dissociation constant values are obtained forProteins #14, #15, and #17 to #22.

TABLE 2 Examples of dissociation constants of designed ankyrin repeatdomains binding to human HGF Protein #* Kd [pM] 23 16 24 163 25 66 26 5127 129 28 26 29 25 *Protein #23 to #29 in this table represent designedankyrin repeat domains consisting of the corresponding amino acidsequence of SEQ ID NO: 23 to 29, and additionally an N-terminal His-tag(SEQ ID NO: 1). Similar HGF dissociation constant values are obtainedfor Proteins #30 to #37.

TABLE 3 Examples of dissociation constants of designed ankyrin repeatdomains binding to human HSA Protein #* Kd [nM] 44 26 45 13 46 22 47 2748 20 49 15 50 27 51 14 52 6 54 11 55 15 *Protein #44, to #55 in thistable represent designed ankyrin repeat domains consisting of thecorresponding amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49,50, 51, 52, 54, and 55, and additionally an N-terminal His-tag (SEQ IDNO: 1). Similar human serum albumin dissociation constant values areobtained for Proteins #40 to #43, #53, #56, and #57.

Example 3 Competition Binding Assays and Receptor Competition BindingAssays

Characterization of designed ankyrin repeat domains with bindingspecificity for VEGF-A, HGF, or serum albumin, respectively, bycompetition assays. Such assays are well known to the person skilled inthe art. For designed ankyrin repeat domains with binding specificity toVEGF-A, a quantitative sandwich enzyme immunoassay technique was usedaccording to the manufacturer (VEGF-A Quantikine kit DVE00, R&DSystems). A monoclonal antibody specific for VEGF-A was pre-coated ontoa microplate. VEGF-A standards and mixtures of VEGF-A (20 pM) andProtein #18, #19, or #20 at varying concentrations were applied to thewells and any free VEGF-A present (i.e. not bound to the designedankyrin repeat domain) is bound by the immobilized antibody. Afterwashing away any unbound substances, an enzyme-linked polyclonalantibody specific for VEGF-A is added to the wells. Following a wash toremove any unbound antibody-enzyme reagent, a substrate solution isadded to the wells and color develops in proportion to the amount ofVEGF-A bound in the initial step. The color development is stopped andthe intensity of the color is measured. In this assay, the testeddesigned ankyrin repeat proteins showed high VEGF-A inhibition potency.IC₅₀ values were calculated from such titration curves obtained asdescribed above using Graph Pad Prism software and standard proceduresknown to the person skilled in the art. For designed ankyrin repeatdomains with binding specificity to HGF, a cMet receptor competitionassay was performed. For that purpose, 5 nM of human cMet receptor inPBS were immobilized on Maxisorp plates overnight at 4° C. After washingwith PBS 0.05% TWEEN 20, the plate was blocked for 2 h with shaking at300 rpm using PBS containing 0.05% TWEEN 20 and 0.25% Casein at RT. Aconstant concentration of 5 nM human HGF was pre-incubated for 30 min atRT with a 1000 nM-1 μM of Proteins #23, #26, #28, and #29 (1:4 dilutionseries each) on a dilution plate in PBS 0.05% TWEEN 20. After washing ofthe ELISA plate with PBS 0.05% TWEEN, pre-incubated samples weretransferred to the ELISA plate and plate was incubated for 2 h at RTwith shaking at 300 rpm. After washing with PBS 0.25% TWEEN, 200 ng/mlanti human HGF antibody was added for 1 h at RT with shaking at 300 rpm.After washing with PBS 0.05% TWEEN, 100 ng/ml HRP conjugated polyclonalanti HGF species antibody was added for 30 min at RT with shaking at 300rpm. Detection was using 1:3 diluted BM blue POD (Roche). Color reactionwas stopped after 15 min by addition of 1 M H2SO4. Readout was done atA450 using A620 as reference wavelength. Example IC₅₀ values obtained bythese assays are given in Tables 4 and 5. Similar VEGF-A IC₅₀ values areobtained with Proteins #12 to #17, #21, and similar HGF IC₅₀ values areobtained with Proteins #24, #25, #27, and #30 to #37.

TABLE 4 Inhibition of the VEGF-A-bindinq of an antibody by designeddomains (mean IC₅₀ values) Protein #* IC₅₀ [pM] #18 13 #19 13 #20  4*Protein #18 to #20 in this table represent designed ankyrin repeatdomains consisting of the corresponding amino acid sequence of SEQ IDNO: 18 to 20, and additionally an N-terminal His-tag (SEQ ID NO: 1).

TABLE 5 Inhibition of HGF bindina to cMET by designed ankyrin repeatdomains (mean IC₅₀ values) Protein #* IC₅₀ [pM] 23  915 26  623 28  95529 1357 *Protein #23, #26, #28, and #29 in this table represent designedankyrin repeat domains consisting of the corresponding amino acidsequence of SEQ ID NO: 23, 26, 28, and 29, and additionally anN-terminal His-tag (SEQ ID NO: 1).

Example 4 Generation of Recombinant Binding Proteins, in ParticularRecombinant Proteins Comprising Two, Three or Four Designed AnkyrinRepeat Domains, and Other Repeat Proteins

DNA encoding designed ankyrin repeat domains or recombinant bindingproteins was generated by genetic means well known to the person skilledin the art. Recombinant binding proteins selected from the group ofamino acid sequences SEQ ID NOs: 58 to 133, additionally having SEQ IDNO: 1 or the amino acids GS at the N terminus or recombinant bindingproteins selected from the group of amino acid sequences SEQ ID NOs: 134to 179, or designed ankyrin repeat domains selected from the group ofamino acid sequences SEQ ID NOs: 10 to 57, additionally having SEQ IDNO: 1 or the amino acids GS at the N terminus, were expressed in thecytoplasm of Escherichia coli using standard techniques using the pQEexpression system from Qiagen (Germany). In case the amino acids GS wereat the N terminus, the Met residue additionally encoded by theexpression vector was efficiently cleaved off in the cytoplasm of E.coli from the expressed polypeptide since the start Met is followed by asmall Gly residue (i.e. the amino acid at position 1 of SEQ ID NOs: 134to 179). The cells were lysed by using a French press, and the proteinswere purified to near homogeneity from the crude cell extract by usingstandard chromatographic techniques well known to the person in the art.

Example 5 Improving Pharmacokinetic Properties with Increasing Numbersof Designed Ankyrin Repeat Domains with Binding Specificity for SerumAlbumin Comprised in a Recombinant Binding Protein—Mouse PharmacokineticStudies

For mouse pharmacokinetic studies Proteins #57, #62, #63, #64, #68, #73,#74, #82, #83, #97, #109, and #110 (proteins corresponding to SEQ IDNOs: 57, 62, 63, 64, 68, 73, 74, 82, 83, 97, 109, and 110, additionallyhaving SEQ ID NO: 1 at the N terminus) prepared as described in Example4, were labeled radioactively as described (Zahnd, C., Kawe, M., Stumpp,M. T., de Pasquale, C., Tamaskovic, R., Nagy-Davidescu, G., Dreier, B.,Schibli, R., Binz, H. K., Waibel, R., Plückthun, A., Cancer Res. 70,1595-1605, 2010) and administered at 10 μg in 100 μl as a singleintravenous bolus injection into the tail vein of female BALB/c mice,respectively. Serum samples from each mouse were collected at varioustime points and the accumulated radioactivity was determined using agamma-scintillation counter.

In these experiments, proteins comprising two designed ankyrin repeatdomains with binding specificity for serum albumin consistentlyexhibited improved pharmacokinetic properties compared to comparableconstructs comprising only one designed ankyrin repeat domain withbinding specificity for serum albumin (FIGS. 3A-3D). For example, thecomparison of Protein #57, which comprises a single designed ankyrinrepeat domain with binding specificity for serum albumin (SEQ ID NO: 57,comprising SEQ ID NO: 51 plus a C-terminal polypeptide) with Proteins#62 and #63, which comprise two designed ankyrin repeat domains withbinding specificity for serum albumin (two times SEQ ID NO: 51, linkedby GS—(SEQ ID NO: 63) or PT-rich (SEQ ID NO: 62) polypeptide linkers)shows that having two designed ankyrin repeat domains with bindingspecificity for serum albumin leads to higher % ID at e.g. 24 h (+57%GS; +59% PT), 48 h (+76% GS; +82% PT) or 72 h (+79% GS; +94% PT)post-injection, and leads to an improved terminal half-life (+38% GS;+48% PT) compared to the protein comprising only a single designedankyrin repeat domain with binding specificity for serum albumin (FIG.3A). In particular, using a PT-rich linker, in particular SEQ ID NO: 9,leads to improved pharmacokinetic properties (FIG. 3A). The followingthree examples show that the method of improving pharmacokineticproperties by having two (instead of one) designed ankyrin repeatdomains with binding specificity for serum albumin present in a protein,is transferable to different proteins comprising different designedankyrin repeat domains. For example, the comparison of thepharmacokinetic profile of Protein #64, comprising SEQ ID NOs: 22(designed ankyrin repeat domain with binding specificity for anothertarget than serum albumin) and 51 (designed ankyrin repeat domain withbinding specificity for serum albumin), with Proteins #73 and #74,comprising each SEQ ID NOs: 22 and two times 51 (Protein #73 has SEQ IDNOs: 51 flanking SEQ ID NO: 22, and Protein #74 has SEQ ID NOs: 51N-terminal of SEQ ID NO: 22) shows that having two designed ankyrinrepeat domains with binding specificity for serum albumin leads tohigher % ID at e.g. 24 h (+62% N-terminal; +89% flanking), or 48 h(+136% N-terminal; +175% flanking) post-injection, and leads to animproved terminal half-life (+>63% for both N-terminal or flanking)compared to the protein comprising only a single designed ankyrin repeatdomain with binding specificity for serum albumin (FIG. 3B). Likewise, apharmacokinetic profile comparison of Protein #82, comprising twice SEQID NO: 11 (designed ankyrin repeat domain with no known bindingspecificity) and once SEQ ID NO: 51 (designed ankyrin repeat domain withbinding specificity for serum albumin), with Protein #109, comprisingtwice SEQ ID NO: 11 and twice SEQ ID NO: 51 (N-terminal), indicates thathaving two designed ankyrin repeat domains with binding specificity forserum albumin leads to higher % ID at e.g. 24 h (+12%), or 48 h (+35%)post-injection, and leads to an improved terminal half-life (+71%)compared to the protein comprising only a single designed ankyrin repeatdomain with binding specificity for serum albumin (FIG. 3C).Furthermore, the pharmacokinetic profile comparison of Protein #83,comprising SEQ ID NOs: 38 and 39 (designed ankyrin repeat domains eachwith binding specificity for another target than serum albumin) and 50(designed ankyrin repeat domain with binding specificity for serumalbumin), with Proteins #110, comprising each SEQ ID NOs: 38 and 39 andtwice SEQ ID NO: 50 (flanking SEQ ID NOs: 38 and 39), shows that havingtwo designed ankyrin repeat domains with binding specificity for serumalbumin leads to higher % ID at e.g. 24 h (+198%), 48 h (+198%), or 72 h(+228%) post-injection, and leads to an improved terminal half-life(+19%) compared to the protein comprising only a single designed ankyrinrepeat domain with binding specificity for serum albumin (FIG. 3D).Furthermore, Protein #97 exhibited significantly improvedpharmacokinetic properties over Protein #68, e.g. the terminalhalf-lives were 21 and 16 hours, respectively, indicating that havingtwo designed ankyrin repeat domains with binding specificity for serumalbumin is beneficial over having only one such domain.

These results indicate, surprisingly, that using two designed ankyrinrepeat domains with binding specificity for serum albumin instead of onein a recombinant binding protein leads to improved pharmacokineticproperties, as discussed further in Example 6.

Example 6 Improving Pharmacokinetic Properties with Increasing Numbersof Designed Ankyrin Repeat Domains with Binding Specificity for SerumAlbumin Comprised in a Recombinant Binding Protein—Cynomolgus MonkeyPharmacokinetic Studies

For cynomolgus monkey pharmacokinetic studies Proteins #57, #62, and #97(proteins corresponding to SEQ ID NOs: 57, 62, and 97, additionallyhaving SEQ ID NO: 1 at the N terminus) and Protein #134 (a proteincorresponding to SEQ ID NOs: 134), prepared as described in Example 4,were administered via intravenous infusion for 30 min at a target doselevel of between 0.5-100 mg/kg to cynomolgous monkeys. Blood sampleswere collected pre-dose and again at selected time points, for example 5min, 10 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h,120 h and 168 h post-end of infusion (i.e. post-injection). The bloodsamples were allowed to stand at room temperature and were centrifugedto generate serum, followed by storage at −80° C. pending analyses.Pharmacokinetic parameters were determined using procedures well knownto the person skilled in the art. Serum concentrations of Proteins #57,#62, #97 and #134 were determined by sandwich ELISA using a rabbitmonoclonal anti-designed ankyrin repeat domain antibody as capturereagent and murine monoclonal anti-designed ankyrin repeat domainantibody as detection reagent, and using a standard curve.Pharmacokinetic parameters were determined using standard software suchas Phoenix WinNonLin (Certara, Princeton, USA) or GraphPadPrism(GraphPad Software, La Jolla, USA) and standard analyses such asnon-compartmental analyses. The resulting pharmacokinetic profiles areshown in FIGS. 4A-4B. Proteins comprising two designed ankyrin repeatdomains with binding specificity for serum albumin consistentlyexhibited improved pharmacokinetic properties compared to comparableconstructs comprising only one designed ankyrin repeat domain withbinding specificity for serum albumin. For example by comparing Protein#57 (0.5 mg/Kg; 27.7 nmol/kg), comprising a single designed ankyrinrepeat domain with binding specificity for serum albumin (SEQ ID NO:51), with Protein #62 (1.04 mg/Kg; 34.5 nmol/kg), a protein comprisingtwo designed ankyrin repeat domains with binding specificity for serumalbumin (twice SEQ ID NO: 51, linked by a PT-rich polypeptide linker),the results show that having two designed ankyrin repeat domains withbinding specificity for serum albumin in a protein leads to higherexposure (2138 d*nmol/L vs. 4676 d*nmol/L, i.e. +119% calculated up today 7), leads to a reduced clearance (0.0108 L/(d*kg) vs. 0.0031L/(d*kg); i.e. −71%), and leads to an improved terminal half-life (4.57d vs. 9.00 d, i.e. +97% calculated from day 1 to day 7) compared to theprotein comprising only a single designed ankyrin repeat domain withbinding specificity for serum albumin (FIG. 4A). Also, the percentageinjected dose, normalized to the concentration measured 10 minpost-injection, is increased comparing Protein #57 with Protein #62 atday 4 (23.39% vs. 57.72%; +148%), day 5 (19.00% vs. 48.41%; +155%), andday 6 (18.5% vs. 51.94%; +175%). As a further example in cynomolgusmonkey, Protein #134 (a protein corresponding to SEQ ID NOs: 134,produced as descried in Example 4, was tested at different doses in 10animals each (5 male, 5 female, each dose) and the terminal half-lifewas evaluated using WinNonLin considering concentration values up to day7. Protein #134 exhibited an average terminal half-life of 4.0 days (95h) when given at 1 mg/kg (compared to Protein #97 exhibiting 2.7 days(65 h) terminal half-life at 1 mg/kg (+46%)), 5.3 days (127 h) whengiven at 10 mg/kg, and 5.8 days (139 h) when given at 100 mg/kg tocynomolgus monkey. The pharmacokinetic profile of Protein #134 incomparison to Protein #97 in cynomolgus monkey is shown in FIG. 4B. Likein Example 5, these results indicate, surprisingly, that using twodesigned ankyrin repeat domains with binding specificity for serumalbumin instead of one in a recombinant binding protein leads toimproved pharmacokinetic properties. These results are discussed in thefollowing.

In the absence of any albumin binding activity, a recombinant bindingprotein has a terminal half-life in the range of minutes both in mouseand cynomolgus monkey (See WO 2012/069654). Proteins comprising at leastone designed ankyrin repeat domain with binding specificity for serumalbumin show terminal half-lives which are far greater than if nodesigned ankyrin repeat domain with binding specificity for serumalbumin is present. A pharmacokinetic profile of a protein comprisingone designed ankyrin repeat domain with binding specificity for serumalbumin are shown in FIG. 3A and FIG. 4A.

The art contains a study, in which the effect of valency of anotherserum albumin binding protein domain, the albumin binding domain (ABD)derived from streptococcal protein G, was investigated (Hopp et al.,2010; loc. cit.). Another study uses C-terminally fused peptides (WO2011/095545), which are no protein domains. ABD is a helical proteindomain with binding specificity for serum albumin. Importantly, Hopp etal. 2010 (loc. cit.) show that having two such ABDs (one N-terminal andone C-terminal) in a recombinant binding protein does not lead to asignificantly improved terminal half-life in mouse compared to arecombinant binding protein comprising only one ABD (C-terminal;37.9±1.1 h vs. 36.4±4.8 h). In particular, at 24 h and 72 h postinjection, the recombinant binding protein comprising one ABD showedidentical percentage injected doses as the recombinant binding proteincomprising two ABD, indicating equivalent pharmacokinetic properties ofthe two recombinant binding proteins. Based on the findings with ABD,one skilled in the art would expect that a recombinant binding proteincomprising two albumin binding protein domains such as designed ankyrinrepeat domains with binding specificity for serum albumin would not haveimproved pharmacokinetic properties compared to a recombinant bindingprotein comprising only one designed ankyrin repeat domain with bindingspecificity for serum albumin. Surprisingly, we found that this is notthe case. In contrast to Hopp et al. (loc. cit.), recombinant bindingproteins comprising two designed ankyrin repeat domains with bindingspecificity for serum albumin surprisingly exhibited clearly prolongedterminal half-lives compared to recombinant binding proteins comprisingonly one designed ankyrin repeat domain with binding specificity forserum albumin.

These examples illustrate a number of additional findings. For example,the pharmacokinetic properties of Protein #134 are superior to the onesof Protein #97, illustrating the importance of the choice of theindividual designed ankyrin repeat domains. SEQ ID NO: 134 was chosen tobe composed of components that lead to maximal activity and optimalpharmacokinetic properties. Also, the arrangement of the designedankyrin repeat domains within Protein #134 was chosen to lead to optimalpharmacokinetic properties. When analyzing recombinant binding proteinscomprising four designed ankyrin repeat domains including two designedankyrin repeat domains with binding specificity for serum albuminregarding mouse and cynomolgus monkey pharmacokinetics, the mostfavorable pharmacokinetic properties were observed for recombinantbinding proteins having the two designed ankyrin repeat domains withbinding specificity for serum albumin flanking the other two designedankyrin repeat domains.

As the examples of this example comprise different combinations ofdesigned ankyrin repeat domains with binding specificity for anothertarget than serum albumin, the approach of using at least two designedankyrin repeat domains with binding specificity for serum albumin forimproving the pharmacokinetic properties appears to be generallyapplicable to proteins comprising several designed ankyrin repeatdomains.

Example 7 Simultaneous Binding of Two Human Serum Albumin Molecules byProtein #134

Protein #134 (A recombinant binding protein consisting of SEQ ID NO:134, additionally having GS at the N terminus) was prepared as describedin Example 4. Protein #134, purified human serum albumin (HSA), as wellas Protein #134/HSA mixture (1:2 stoichiometry) were analyzed by sizeexclusion chromatography coupled to multi-angle static light scattering(SEC-MALS). SEC-MALS was performed using the proteins of Table 6 at 30μM (Protein #134) or 60 μM (HSA) concentration on a Agilent 1200 system(Life Technologies, USA) connected to a Wyatt (USA) MALS and refractiveindex detector (flow rate: 0.6 ml/min; injection volume: 100 μl; column:GE Healthcare (USA) Superdex200 10/300 GL). The Protein #134/HSA mixturewas pre-incubated for 3 hours at 20° C. prior injection. Thechromatograms are shown in FIG. 5 and the molecular masses of theeluates were determined and compared to the theoretical molecularmasses, as shown in Table 6. For this experiment, 100 mg HSA (CSLBehring 20% solution) were purified using a Superdex 200_26.60 column ona AEKTA prime system (GE Healthcare; 2.0 ml/min, PBS, isocratic flow,injection volume 10 ml of 1:20 in PBS diluted HSA, collecting 4 mlfractions). The peak fraction of the main peak was used for performanceof the SEC-MALS experiment.

Protein #134 at 30 μM was monodisperse with the elution fractionscontaining protein of the expected molecular weight (FIG. 5). Likewise,purified HSA at 60 μM was monodisperse with the elution fractionscontaining protein of the expected molecular weight (Table 6). Themixture of 30 μM Protein #134 and 60 μM HSA resulted in two peaks inSEC. One peak contained protein complexes of a molecular weightcorresponding to a 1:2 (Protein #134/HSA) complex, indicating that thetwo designed ankyrin repeat domains with binding specificity for serumalbumin are functional simultaneously. Additionally, in the tail of thispeak, protein complexes of a molecular weight corresponding to a 1:1(Protein #134/HSA) complex could be detected. Additionally, free HSAcould be detected. As this amount is minor, one can rule out that themain peak is a 2:1 (Protein #134/HSA) complex, which would theoreticallybe consistent with the observed weight, yet a large fractioncorresponding to 75% of free HSA would be expected. No free Protein#134/HSA could be detected. No peaks with a molecular weightcorresponding larger than the one of the 1:2 (Protein #134/HSA) complexwere detected. SEC-MALS measurements and the variations observed inSEC-MALS measurements are well known to the person skilled in the art.

TABLE 6 Size-exclusion chromatoqraphy coupled to static liqht scatteringof Protein #134 and HSA, as well as the complex Protein #134/HSA.Theoretical MW Peaks stoichiometry & MW measured HSA 69366.6 Da  63350Da Protein #134 62397.0 Da  58700 Da Protein #134/HSA tail 1:1, 131763.6Da 132500 Da Protein #134/HSA middle mixture of 1:1 & 1:2, 173700 Da MWdepends on ratio Protein #134/HSA front 1:2, 201130.2 Da 197500 Da

The simultaneous binding of two human serum albumin molecules by onerecombinant binding protein is similarly observed when analyzing Protein#97, and Protein #102, Protein #109, Protein #110 in size exclusionchromatography coupled to static light scattering.

Example 8 Maximizing Target Binding Activity by Choosing LinkerComposition and by Choosing the Number of Designed Ankyrin RepeatDomains with Binding Specificity for Serum Albumin

Polypeptide linkers that link protein domains are well-known to theperson skilled in the art. Gly-Ser-rich linkers are well-known fromsingle-chain Fv antibody fragments, where they are used to link the twoFv polypeptide chains. Various other polypeptide linkers exist,including e.g. antibody hinge regions, or unstructured polypeptides suchas sequences comprising mostly the amino acids Ala, Glu, Lys, Pro, Ser,Thr (WO 2007/103515) or Ala, Pro, and Ser (WO 2008/155134). Furthermore,Pro-Thr-rich linkers have been disclosed (WO 2014/191574). The effect ofsuch a linker on the properties of protein domains linked by such linkerneeds to be assessed for every linker/domain combination. Next to thenature of a polypeptide linker, we surprisingly found that the number ofserum albumin-binding domains can influence the functionality of aprotein. To maximize target binding activity of the recombinant bindingproteins of the present invention, recombinant binding proteinscomprising Gly-Ser-rich and Pro-Thr-rich polypeptide linkers werecompared as well as recombinant binding proteins comprising one or twodesigned ankyrin repeat domains with binding specificity for serumalbumin. For that purpose, Proteins #69, #71, and, #107, eachadditionally having SEQ ID NO: 1 at the N terminus and prepared asdescribed in Example 4, were analyzed for binding to VEGF-A and HGF,respectively, by ELISA (for methods see Example 4). The results areshown in Table 7. The comparison of the EC₅₀ values of Protein #69 withProtein #71 indicates that the recombinant binding protein withPro-Thr-rich linkers is more potent with respect to binding of VEGF-A(factor 2) and HGF (factor 1.3), respectively, compared to therecombinant binding proteins with Gly-Ser-rich linkers. The comparisonof the EC₅₀ values of Protein #69 with Protein #107 indicates that therecombinant binding protein comprising two designed ankyrin repeatdomains with binding specificity for serum albumin is more potent withrespect to binding of VEGF-A (factor 1.4) and HGF (factor 1.1),respectively, compared to the recombinant binding protein comprisingonly one designed ankyrin repeat domain with binding specificity forserum albumin. This is surprising in view of previous results (Hopp etal., 2010), where the presence of two albumin binding domains in aconstruct have a negative impact on the functionality of the molecule.This result indicates that recombinant binding proteins preferentiallycomprises Pro-Thr-rich linkers and two designed ankyrin repeat domainswith binding specificity for serum albumin, rather than Gly-Ser-richlinkers and one designed ankyrin repeat domains with binding specificityfor serum albumin.

TABLE 7 ELISA analysis of recombinant binding proteins with differentlinkers and different numbers of designed ankyrin repeat domains withbinding specificity for serum albumin Protein Number of EC₅₀ [nM] EC₅₀[nM] #* Linker SABD† VEGF-A HGF  69 PT 1 0.102 0.117  71 GS 1 0.2050.179 107 PT 2 0.073 0.107 *Protein #69, #71, and #107 in this tablerepresent designed ankyrin repeat domains consisting of thecorresponding amino acid sequence of SEQ ID NOs: 69, 71, and 107, andadditionally an N-terminal His-tag (SEQ ID NO: 1). †Number of designedankyrin repeat domains with binding specificity for serum albumin

Example 9 Improvement of Protein Stability when Using SEQ ID NO: 50

Proteins #48, #49, and #51 were further characterized for their midpointof denaturation temperature (i.e. midpoint of the cooperative unfoldingupon temperature increase) by mixing the Proteins (25 μl; 100 μM in PBS)with a fluorescent dye (25 μl Sypro orange (Life Technologies) diluted1/2500 in PBS) and measuring a melting curve with a thermal cyclercomprising a fluorescence reader (CFX96 Real-Time PCR Detection System;Biorad; 25 seconds holding time every 0.5° C. followed by fluorescenceread), essentially as described by Niesen et al. 2007 (Niesen, F. H.,Berglund, H., Vedadi, M., Nature Protocols 2, 2212-2221, 2007). In PBS,Protein #48 exhibited a midpoint of denaturation of 83.5° C., andProtein #49 exhibited a midpoint of denaturation of 84.5° C., whileProtein #51 exhibited a midpoint of denaturation of 79.5° C.

In order to identify the designed ankyrin repeat domain with bindingspecificity for serum albumin having the best storage stabilityproperties, Proteins #49, #50 and #51 (corresponding to SEQ ID NOs: 49,50 and 51, respectively, additionally having SEQ ID NO: 1 at the Nterminus) were prepared as described in Example 4, and samples wereconcentrated to 10 mg/ml in PBS. Proteins #50 and #51 where then storedfor 1 month at −80° C. or at 40° C. in glass vials, followed by analysison SDS 15% PAGE. While Proteins #50 and #51 showed equivalent stabilityupon storage at −80° C., Proteins #50 showed significantly reducedamounts of degradation products by >50% reduction compared to Protein#51 on SDS 15% PAGE after 1 month storage at 40° C. Similarly, whenstored at 4° C., 25° C., 40° C. and 60° C. for one week at 10 mg/ml inPBS, Protein #50 showed significantly reduced amounts of degradationproducts compared to Protein #49. In particular, Protein #50 showed >50%reduction of degradation products compared to Protein #49 on SDS 15%PAGE both when stored at 40° C. or 60° C., respectively (FIG. 2).

These findings illustrate that Protein #50 has an improved storagestability compared to Proteins #49 and #51. Similarly, when comparingthe storage stability of Proteins #48 to #51 (corresponding to SEQ IDNOs: 48 to 51, additionally having SEQ ID NO: 1 at the N terminus;produced as described in Example 4), by incubating the proteins at 10mg/ml in PBS in glass vials for 1 month at 40° C., Proteins #48 to #50exhibit >30% reduction of degradation products compared to Protein #51.

These findings are corroborated by testing the storage stability ofProtein #102 and Protein #103 (recombinant binding proteins consistingof the amino acid sequences corresponding to SEQ ID NOs: 102 and 103,both additionally having SEQ ID NO: 1 at the N terminus). Protein #102and Protein #103 were prepared as described in Example 4, samples wereconcentrated to 10 mg/ml in PBS and stored for 1 month at −80° C. in orat 40° C. in glass vials, followed by analysis on standardsize-exclusion chromatography. While Protein #102 and #103 showedequivalent elution profiles upon storage at −80° C., Protein #102 showed98.72% monomeric species and Protein #103 showed 100% monomeric speciesupon storage at 40° C. This indicates that having SEQ ID NO: 50 presentin the recombinant binding protein is more favorable regarding storagestability than having SEQ ID NO: 49 present. Similarly, Protein #103exhibits lower amounts of degradation products than Protein #102 whenanalyzed by SDS-PAGE after 1 month storage at 40° C. in glass vials inPBS at 10 mg/ml, confirming the higher storage stability of arecombinant binding protein comprising SEQ ID NO: 50 in comparison tothe recombinant binding protein comprising SEQ ID NO: 49.

Similar results are obtained when comparing Protein #134 with Protein#143 or Protein #150 (recombinant binding proteins consisting of theamino acid sequences corresponding to SEQ ID NOs: 134, 143 and 150,respectively), prepared as described in Example 4. When stored at 40° C.for one month at 10 mg/ml in PBS in glass vials, Protein #134 shows >50%reduction of degradation products compared to Proteins #143 and #150when analyzed on SDS 15% PAGE. This indicates that having SEQ ID NO: 50present in the recombinant binding protein is more favorable regardingstorage stability than having either SEQ ID NO: 49 or SEQ ID NO: 51present.

Example 10 Characterization of Recombinant Binding Proteins Using ELISA

Purified recombinant binding protein consisting of the amino acidsequence SEQ ID NO: 134, prepared as described in Example 4, wassubjected to ELISA analyses. 100 μl or 50 μl of 20 nM target (VEGF-A,HGF, or serum albumin) in PBS per well were immobilized in a Maxisorpplate (Nunc, Denmark) overnight at 4° C. After washing 5 times with 300μl PBST (PBS supplemented with 0.1% Tween 20), the wells were blockedwith 300 μl PBST-C (PBST supplemented with 0.25% casein) for 2 h at roomtemperature with shaking at 450 rpm on a Titramax 1000 shaker (Heidolph,Germany). After washing 5 times as described above, 100 μl/well or 50μl/well Protein #134 (concentrations ranging from 100 nM to 0.01 μM) inPBST-C was applied and incubated for 1 h to 2 h at room temperature withshaking at 450 rpm. After washing 5 times as described above, binding ofProtein #134 was detected using 100 μl or 50 μl/well rabbitanti-designed ankyrin repeat domain monoclonal antibody in PBST-C for 1h at room temperature with shaking at 450 rpm. After washing 5 times asdescribed above, bound anti-designed ankyrin repeat domain antibody wasdetected using 100 μl or 50 μl/well goat anti-rabbit IgG-HRP conjugatein PBST-C for 1 h at room temperature with shaking at 450 rpm. Afterwashing 5 times as described above, the ELISA was then developed using100 μl BM soluble blue POD substrate (Roche, Switzerland), diluted 1:4in water. The reaction was stopped after 5 min using 100 μl 1 M H₂SO₄.The OD (OD 450 nm-OD 620 nm) was then recorded. The ELISA resultsindicate that Protein #134 binds human, cynomolgus monkey, rat and mouseVEGF-A with equivalent potency (Table 8 and FIG. 6A). Cynomolgus monkeyVEGF-A is identical to human VEGF-A and was thus not tested separately.No binding of Protein #134 to VEGF-C and PDGF-AB was detected (Table 9and FIG. 6A). Human, cynomolgus and mouse HGF is bound by Protein #134with equivalent potency (EC₅₀ values in the 20-50 pM range; Table 8 andFIG. 6B). Furthermore, Protein #134 binds serum albumin of human,cynomolgus monkey, rat, dog and mouse with equivalent potency (EC₅₀values in the 10-20 pM range; Table 8 and FIG. 6C). A comparison ofProtein #134 (i.e. protein consisting of SEQ ID NO: 134) with Protein#60 or Protein #61 (i.e. proteins consisting of SEQ ID NOs: 60 or 61,additionally having SEQ ID NO: 1 at the N terminus, produced asdescribed in Example 4), revealed that the EC₅₀ of Protein #134 observedfor the binding of human serum albumin is significantly better than theones observed for Proteins #60 or #61 (225 pM or 322 pM, respectively).

TABLE 8 Apparent EC₅₀ values of Protein #134 for binding VEGF-A HGF andserum albumin of different species EC₅₀ [pM] EC₅₀ [pM] EC₅₀ [pM] VEGF-AHGF (95% SA (95% Species (95% C.I.)* C.I.)* C.I.)* Human 24 (20-27) 24(20-29) 13 (10-16) Mouse 21 (19-23) 45 (38-52) 15 (13-17) Rat 22 (18-26)n.a. 17 (13-21) Dog n.a. n.a. 23 (20-25) Cynomolgus 24 (20-27)† 40(36-44) 17 (13-21) monkey *C.I. confidence interval, †100% sequenceidentity to human VEGF-A, thus the value for human VEGF-A is listed,n.a. not analyzed

TABLE 9 Apparent EC₅₀ values of Protein #134 for bindins different humanVEGFs and PDGFs EC₅₀ [pM] Target (95% C.I.)* Human VEGF-A 24 (20-27)Human VEGF-C No binding detected Human PDGF-AB No binding detected *C.I.confidence interval

Example 11 Characterization of Recombinant Binding Proteins UsingCompetition Assays

Purified recombinant binding protein consisting of the amino acidsequence SEQ ID NO: 134, prepared as described in Example 4, was subjectto competition ELISA and FRET analyses. Such competition FRET and ELISAassays are well known to the person skilled in the art. Protein #134 wasmeasured in a VEGF-A/VEGFR-2 competition FRET assay. For that purpose,Protein #134 and biotinylated VEGF-A165 (Reliatech, #300-076Bi-L) wereprepared as eight-fold concentration stocks in PBS containing 0.2% BSAand 0.01% Tween (PBST-BSA). A competition mixture of 5 μl eight-foldconcentration Protein #134 and 5 μl of eight-fold concentrationbiotinylated VEGF-A165 were pre-incubated for 1 hour at room temperature(competition mixture). In parallel, 5 μl of Streptavidin-Tb(streptavidin-Lumi4-terbium cryptate donor, Cisbio #610SATLB) and 5 μlof PAb anti-hlgG-de (D2-conjugated goat anti-Human IgG, Cisbio #61HFCDAA) were added to 500 μl of PBST-BSA buffer and incubated for 20minutes (2× reagent). Ten μl/well of 2× reagent were dispensed in a384-well HTRF white-plate (Thermo Fisher Scientific Inc.) and 5 μl/wellof four-time concentration hVEGF-R2-Fc fusion (Reliatech #SFC-008) wereadded. Five μl of the preincubated competition mixture were then addedto wells. The complete reaction mix was incubated in the dark for 1 hourat room temperature before the fluorescence read out using afluorescence reader. The final mixture contained 10 nM solubleVEGF-R2-Fc fusion, 10 nM biotinylated VEGF-A, and varying concentrationsof Protein #134. The read-out was done for A665 nm and A595 nmwavelength (Excitation 340 nm). The results of the assay are shown inFIG. 7A. In this assay, Protein #134 inhibits the VEGF-A/VEGFR-2interaction with an IC₅₀ value of 0.6 nM. Protein #134 was furthermeasured in a HGF/cMet competition ELISA experiment as described inExample 3. The results of the assay are shown in FIG. 7B. In this assay,Protein #134 inhibits the HGF/cMet interaction with an IC₅₀ value of0.92 nM. Protein #134 was also measured in a VEGF-A competition ELISAexperiment as described in Example 3. The results are shown in FIG. 7C.In this assay, Protein #134 inhibits VEGF-binding with an IC₅₀ value inthe single-digit pM range (IC₅₀ 4.5 pM).

Example 12 Characterization of Simultaneous Target Binding ofRecombinant Binding Proteins Using Surface Plasmon Resonance

SPR was measured in a similar way as described in Example 2, with thefollowing setup. 2700 RU human HGF were immobilized on a sensor chip.Then 100 nM Protein #134 or PBST were injected for 180 seconds followedby a PBST wash of 360 seconds. Following this, 100 nM human VEGF-A orPBST were injected for 180 seconds (leading to saturation) followed by aPBST wash of 360 seconds. Finally, 100 nM human serum albumin or PBSTwere injected for 180 seconds followed by a PBST wash of 600 seconds.The resulting signals are shown in FIG. 8. The results indicate thatProtein #134 can bind HGF, VEGF-A, and serum albumin. Furthermore, theresults indicate that Protein #134 can bind HGF and VEGF-A, as well asHGF, VEGF-A, and serum albumin at the same time.

Example 13 Characterization of Recombinant Binding Proteins in CellCulture

Purified recombinant binding protein consisting of the amino acidsequence SEQ ID NO: 134, prepared as described in Example 4, was furthersubjected to cellular assays including a HUVEC proliferation assay toassess VEGF-A inhibition, and an A549 cell migration assay as well as acMet phosphorylation assay, both to assess HGF inhibition, assays wellknown to the person in the art.

Inhibition of VEGF-A-induced HUVEC proliferation was determined bytitrating increasing Protein #134 concentrations in the HUVECproliferation assay. Human VEGF-A was used at a concentration of 8 ng/ml(corresponding to EC80 as determined in a proliferation assay). Protein#134 was titrated from 200 ng/ml to 0.195 ng/ml. Cells were seeded in 50μl assay medium. Protein dilutions (in assay medium) were made by serialdilution 1:2 fold in a dilution plate; the concentration was four timesthe final concentration. Protein #134 dilutions were mixed withfour-fold VEGF-A concentrations (32 ng/ml; final 8 ng/ml) in a ratio1:1. 50 μl of the mixtures were added to the cells for 72 h. Cellproliferation was determined either by BrdU incorporation in thereplicating DNA or by monitoring metabolic activity using WST-1. Theresults are shown in FIG. 9A, indicating that Protein #134 exhibits anIC₅₀ of 5.7 ng/ml (91.35 μM).

Inhibition of the HGF/cMet interaction was determined using Protein #134in an Oris cell migration assay (Platypus Technologies, USA). The assaywas performed according to the manufacturers' protocol. Briefly, cellswere seeded with 100,000 A549 cells in serum-free DMEM. Cells adheredafter 24 hours and medium was exchanged to assay medium; DMEM with andwithout 0.5 nM HGF with and without 5 μM Protein. HGF and theneutralizing Protein were preincubated for 1 h at RT before addition tocells. The Oris™ stoppers were removed. The assay was then incubated for48 hours to permit cell migration. Cells were stained with Calcein (2.5ng/ml) for 40 minutes and images were taken. The migration zone wasmeasured using an inverse microscope Olympus and its software CellSensDimension. The migration area was calculated as the covered area bysubtracting the uncovered area of the pre-migration well with theuncovered area of respective samples wells. The uncovered area was thecell-free area and was measured using the diameter function in theprocessing folder of the software. The results are shown in FIG. 9B andindicate that Protein #134 can suppress the HGF-induced cell migrationof A549 cells.

Inhibition of cMet phosphorylation by Protein #134 was measured usingA549 cells and a DuoSet P-cMet-ELISA (RnD Systems). Cells were seeded incomplete medium in 96 well plates ad 200.000 cells per well in completemedium. 24 h later medium was replaced by serum-free medium. Cells wereincubated for another 24 h and stimulated by 1 nM human HGF (or PBS fornegative control) in the presence and absence of Protein #134. HGF andProtein #134 were preincubated for at least 30 min at room temperatureprior to addition to cells. Cells were stimulated for 10 minutes at roomtemperature. Stimulation was terminated by removing the cell supernatant(by flicking) and addition of cell lysis buffer according to protocol.Cell lysates were kept at −20° C. until the ELISA experiment. Theresults are shown in FIG. 9C and indicate that Protein #134 can suppressHGF-mediated cMet phosphorylation with an IC₅₀ of 184 μM.

Example 14 Effect of Recombinant Binding Proteins on Tumor Growth InVivo

A U87MG xenograft mouse model was used to assess the benefit of having adesigned ankyrin repeat domain with binding specificity for VEGF-Acombined with a designed ankyrin repeat domain with binding specificityfor HGF compared to having them separate. Protein #134 consisting of SEQID NO: 134 (comprising two designed ankyrin repeat domains with bindingspecificity for serum albumin each consisting of amino acids of SEQ IDNO: 50, comprising one designed ankyrin repeat domain with bindingspecificity for VEGF-A consisting of SEQ ID NO: 18, and comprising onedesigned ankyrin repeat domain with binding specificity for HGFconsisting of amino acids of SEQ ID NO: 26), Protein #61 consisting ofSEQ ID NO: 61 (comprising a designed ankyrin repeat domain with bindingspecificity for serum albumin, consisting of amino acids of SEQ ID NO:50, and comprising a designed ankyrin repeat domain with bindingspecificity for VEGF-A, consisting of amino acids of SEQ ID NO: 18) andadditionally having SEQ ID NO: 1 at the N-terminus, or Protein #60consisting of SEQ ID NO: 60 (comprising a designed ankyrin repeat domainwith binding specificity for serum albumin, consisting of amino acids ofSEQ ID NO: 50, and comprising a designed ankyrin repeat domain withbinding specificity for HGF, consisting of amino acids of SEQ ID NO: 26)and additionally having SEQ ID NO: 1 at the N-terminus, were prepared asdescribed in Example 4. For the in vivo analysis, 21*0⁶ U87MG cells permouse were implanted subcutaneously into the right flank of female NMRInu/nu mice (Harlan) and the mice were grouped in groups with equivalenttumor volumes each. On day 29 and day 32, mice were treated with PBS or4 mg/kg protein i.v. On day 35, tumors were harvested and cryo-frozen.On every day of treatment the tumor volume of each tumor was measuredusing the formula: Volume=(width)²×length/2. Body weight measurementsindicated no significant difference between the four treatment groups.Tumor cross-sections were then stained using an antibody for Ki67(ab66155; Abcam, U.K.) for the quantification of proliferation, or usingan antibody for CD-31 (BD550274; BD Biosciences, USA) for thequantification of angiogenesis using standard IHC methods. Thepercentage of proliferative cells and the percentage of mean vascularareas were measured using the software Image J. The results are shown inFIG. 10A. Compared to the PBS, Protein #60 as well as Protein #61inhibit proliferation, and Protein #60 (slightly) as well as Protein #61inhibit angiogenesis, as expected. The combination of the two, resultingin Protein #134, however, leads to improved inhibition of bothproliferation and angiogenesis. This indicates that the combination ofanti-VEGF-A and anti-HGF activity is key for good efficacy.

Protein #134 was further characterized in two patient-derived xenograftmouse models, a gastric cancer model and a renal cancer model.Patient-derived tumor xenograft mouse models are well known to theperson skilled in the art. Protein #134 was prepared as described inExample 4.

For the renal cancer patient-derived xenograft mouse model, renal cellcancer specimens from surgical specimens were implanted s.c. in NMRInu/nu mice and passaged three to five times until establishment ofstable growth patterns. After removal from donor mice, tumors were cutinto fragments of 4-5 mm diameter, which were implanted s.c. in NMRInu/nu mice. Upon obvious onset of solid tumor growth, mice wererandomized to groups of three animals each, and test articles wereadministered as follows to one animal group each: PBS was given i.v. at10 ml/kg three times weekly for three times; Protein #134 was given i.v.at 4 mg/kg three times weekly for three times; sorafenib was given p.o.at 200 mg/kg daily for 21 days. Tumor volumes were assessed as describedabove at the day of treatment start as well as on days 3, 7, 10, 14, 18,and 21. The results are shown in FIG. 10B. In this model, Protein #134is more efficacious than sorafenib, today's standard of care for thetreatment of renal cell carcinoma.

For the gastric cancer patient-derived xenograft mouse model, gastriccancer specimens from surgical specimens were implanted to in NMRI nu/numice and passaged three to five times until establishment of stablegrowth patterns. After removal from donor mice, tumors were cut intofragments of 4-5 mm diameter, which were implanted s.c. in NMRI nu/numice. Upon obvious onset of solid tumor growth, mice were randomized togroups of eight animals each, and test articles were administered asfollows to one animal group each: PBS was given i.v. at 10 ml/kg on days0, 3, 6, 9, 12, 15, and 18; Protein #134 was given i.v. at 4 ml/kg ondays 0, 3, 6, 9, 12, 15, and 18; Paclitaxel was given i.v. at 15 mg/kgon days 0, 7, and 14; Protein #134 plus paclitaxel were given i.v. at 4mg/kg on days 0, 3, 6, 9, 12, 15, and 18, and i.v. at 15 mg/kg on days0, 7, and 14. Tumor volumes were assessed as described above at the dayof treatment start as well as on days 2, 6, 13, 16, and 20. The resultsare shown in FIG. 10C. In this model, Protein #134 was at least asefficacious as paclitaxel, and a combination of the two wassignificantly more efficacious than the individual components.

The invention claimed is:
 1. A recombinant binding protein comprisingthe amino acid sequence of SEQ ID NO:
 134. 2. The binding protein ofclaim 1, wherein the binding protein has binding specificity for HGF,VEGF-A, and serum albumin.
 3. The binding protein of claim 1, whereinthe binding protein is capable of binding to HGF, VEGF-A, and serumalbumin simultaneously.
 4. A pharmaceutical composition comprising thebinding protein of claim 1 and a pharmaceutically acceptable carrierand/or diluent.
 5. A method of treating a medical condition, the methodcomprising the step of administering to a patient in need thereof atherapeutically effective amount of a recombinant binding proteincomprising the amino acid sequence of SEQ ID NO: 134, wherein themedical condition is a neoplastic disease, pathological angiogenesis,and/or an inflammatory disorder.
 6. The method of claim 5, wherein themedical condition is renal cancer.
 7. The method of claim 5, wherein themedical condition is gastric cancer.
 8. The method of claim 5, whereinthe medical condition is multiple myeloma.
 9. A recombinant bindingprotein comprising at least one designed ankyrin repeat domain, whereinthe binding protein has binding specificity for VEGF-A, and wherein thebinding protein comprises the amino acid sequence of SEQ ID NO:
 134. 10.The binding protein of claim 9, wherein the VEGF-A is human VEGF-A. 11.The binding protein of claim 9, wherein the binding protein inhibitsbinding interaction between VEGF-A and VEGFR-2.
 12. The binding proteinof claim 9, wherein the binding protein inhibits VEGF-A-inducedproliferation of HUVEC cells.
 13. A pharmaceutical compositioncomprising the binding protein of claim 9 and a pharmaceuticallyacceptable carrier and/or diluent.
 14. A method of treating a medicalcondition, the method comprising the step of administering to a patientin need thereof a therapeutically effective amount of a recombinantbinding protein comprising at least one designed ankyrin repeat domain,wherein the binding protein has binding specificity for VEGF-A, whereinthe binding protein comprises the amino acid sequence of SEQ ID NO: 134,and wherein the medical condition is a neoplastic disease, pathologicalangiogenesis, and/or an inflammatory disorder.
 15. A recombinant bindingprotein comprising at least one designed ankyrin repeat domain, whereinthe binding protein has binding specificity for HGF, and wherein thebinding protein comprises the amino acid sequence of SEQ ID NO:
 134. 16.The binding protein of claim 15, wherein the HGF is human HGF.
 17. Thebinding protein of claim 15, wherein the binding protein inhibitsbinding interaction between HGF and cMet.
 18. The binding protein ofclaim 15, wherein the binding protein inhibits HGF-induced cellmigration of A549 cells.
 19. The binding protein of claim 15, whereinthe binding protein inhibits HGF-induced phosphorylation of cMet in A549cells.
 20. A pharmaceutical composition comprising the binding proteinof claim 15 and a pharmaceutically acceptable carrier and/or diluent.21. A method of treating a medical condition, the method comprising thestep of administering to a patient in need thereof a therapeuticallyeffective amount of a recombinant binding protein comprising at leastone designed ankyrin repeat domain, wherein the binding protein hasbinding specificity for HGF, wherein the binding protein comprises theamino acid sequence of SEQ ID NO: 134, and wherein the medical conditionis a neoplastic disease, pathological angiogenesis, and/or aninflammatory disorder.
 22. A binding protein consisting of the aminoacid sequence of SEQ ID NO:
 134. 23. A pharmaceutical compositioncomprising the binding protein of claim 22 and a pharmaceuticallyacceptable carrier and/or diluent.
 24. A method of treating a medicalcondition, the method comprising the step of administering to a patientin need thereof a therapeutically effective amount of a recombinantbinding protein consisting of the amino acid sequence of SEQ ID NO: 134,wherein the medical condition is a neoplastic disease, pathologicalangiogenesis, and/or an inflammatory disorder.
 25. The method of claim24, wherein the medical condition is renal cancer.
 26. The method ofclaim 24, wherein the medical condition is gastric cancer.
 27. Themethod of claim 24, wherein the medical condition is multiple myeloma.